Human monoclonal antibody against a costimulatory signal transduction molecule AILIM and pharmaceutical use thereof

ABSTRACT

Immunization of human antibody-producing transgenic mice, which have been created using genetic engineering techniques, with AILIM molecule as an antigen resulted in various human monoclonal antibodies capable of binding to AILIM and capable of controlling a variety of biological reactions (for example, cell proliferation, cytokine production, immune cytolysis, cell death, induction of ADCC, etc.) associated with AILIM-mediated costimulatory signal (secondary signal) transduction. Furthermore, it has been revealed that the human monoclonal antibody is effective to treat and prevent various diseases associated with AILIM-mediated costimulatory signal transduction, being capable of inhibiting the onset and/or advancement of the diseases.

[0001] This application claims priority from Japanese Application No.2000-147116, filed May 18, 2000, and Japanese Application No.2001-99508, filed Mar. 30, 2001.

FIELD OF THE INVENTION

[0002] The present invention relates to human antibodies which bind toAILIM (activation inducible lymphocyte immunomodulatory molecule, alsoreferred to as ICOS (inducible co-stimulator); human monoclonalantibodies which bind to AILIM or a portion thereof; DNA encoding saidhuman monoclonal antibody or a portion thereof, or a portion of saidDNA; cells (including genetic recombinant cells) producing said humanmonoclonal antibody or a portion thereof; human monoclonal antibody or aportion thereof produced by said genetic recombinant cells;pharmaceutical composition comprising said human monoclonal antibody ora portion thereof; pharmaceutical composition comprising antibody toAILIM for treating disorders related to the delayed allergy; method foridentifying, quantitating or assaying substances that bind to AILIM orAILIM ligand; and kit used for said method.

BACKGROUND OF THE INVENTION

[0003] A living body of mammals has immune response systems thatexcludes pathogenic microorganisms (viruses, bacteria, parasites, etc.)or foreign bodies (both are called “antigen” in the following) that haveinvaded the living body. One of them is called natural immune responsesystem, another acquired immune response system. The former is anexclusion mechanism comprising phagocytosis by phagocytes(polymorphonuclear leukocytes, monocytes, macrophages, etc.), attack bynatural killer (NK) cells, and non-specific recognition such asopsonization of antigen by complements. The latter, acquired immuneresponse system, is an exclusion mechanism by lymphocytes (mainly, Tcells and B cells) that acquired the specificity to the antigen (namely,activated lymphocytes). B cells that acquired antigen specificity attackthe antigen existing outside of the cells through production ofantibodies specific to the antigen. T cells that acquired antigenspecificity (namely, activated T cells) are classified into helper Tcells and cytotoxic T cells (cytotoxic lymphocyte, CTL). The helper Tcells regulate a differentiation of B cells and a production ofantibodies, and destroy the antigen cooperating with phagocytes. Thelatter, CTLs attack virus-infected cells and so on by themselves(Experimental Medicine: SUPPLEMENT, “Bio Science Term Library,Immunity”, Yodosha, pp.14-17 (1995)).

[0004] This acquisition of antigen specificity by T cells (namely,activation of T cells) is initiated through recognition by T cells theantigen presented by antigen-presenting cells (APC) such as macrophage,B cells, or dendritic cells. Antigen-presenting cells process theantigens so incorporated and present these processed antigens throughbinding them to major histocompatibility complex (MHC). T cells receiveprimary signal for activation of the cells (or acquisition ofspecificity) by recognizing the processed antigens presented byantigen-presenting cells through a complex between T cell receptor (TcR)and CD3 antigen existing on the surface of the cell membrane (TcR/CD3complex).

[0005] However, the TcR/CD3 complex-mediated primary signal alone cannotactivate T cells sufficiently and leads to unresponsiveness or clonalanergy, so that the cells can not react with any stimulation receivedthereafter. The autocrine of interleukin 2 (IL-2) is necessary for Tcells to be activated, to be differentiated into antigen specific T cellclones, and to be proliferated. In clonal anergy, T cells areinactivated due to no production of IL-2 and such and no cell division.Namely, the activation of T cells accompanied by production of cytokinessuch as IL-2 requires the secondary signal following the first signalthrough TcR/CD3 complex. This secondary signal is called costimulatorysignal.

[0006] T cells receive this secondary signal and transmit it into thecells by interacting (cell adhesion) with molecules other than MHC onantigen-presenting cells through molecules other than TcR/CD3 complex onthe T cell surface. This secondary signal avoids cell anergy (clonalanergy) and activates the cells.

[0007] Although some part of the mechanism of the secondary signaltransmission between antigen-presenting cells and lymphocytes such as Tcells have not yet been elucidated in detail, studies so far haverevealed that an important factor for the secondary signal transmissionis the interaction of CD28 (also named Tp44, T44, or 9.3 antigen), whichis a cell surface molecule expressed mainly on T cells and thymus cells,with CD80 (also named B7-1, B7, BB1, or B7/BB1), which is a cell surfacemolecule expressed on antigen-presenting cells (macrophages, monocytes,dendritic cells, etc.) and with CD86 (also named B7-2 or B70), which isalso a cell surface molecule on antigen-presenting cells (namely, celladhesion through the binding between these molecules). Moreover, it hasbeen experimentally elucidated that the interaction of Cytolytic Tlymphocyte associated antigen 4 (CTLA-4), whose expression is thought tobe enhanced depending on the secondary signal, with the CD80 (B7-1) andCD86 (B7-2) (namely. cell adhesion through the binding between thesemolecules) also plays an important role in the regulation of T cellactivation by the secondary signal. In other words, the regulation of Tcell activation by the transmission of the secondary signal involves atleast the interaction between CD28 and CD80/CD86, the enhancement ofCTLA-4 expression, which is thought to depend on the interaction, andthe interaction between CTLA-4 and CD80/CD86.

[0008] CD28 is known to be a costimulator molecule transmitting thesecondary signal (costimulatory signal) required for the activation of Tcells and for the avoidance of anergy. The secondary signal transmittedby binding this molecule to costimulator molecules, CD80 (B7-1) and CD86(B7-2), on antigen-presenting cells (cell adhesion through the bindingbetween these molecules), stabilizes mRNA of Th1-type cytokines andconsequently promotes production by T cells of a large amount ofTh1-type cytokines such as IL-2, IFNγ, and TNFα. The expression ofCTLA-4 is induced by the primary signal transmitted through TcR/CD3, andthe expression is also enhanced by the secondary signal transmitted bythe binding between CD28 and CD80. It is being revealed that CTLA-4receives these signals to work to inhibit T cell function, which iscontrary to the activation of T cells by the secondary signaltransmitted by CD28.

[0009] Human CD28 and CTLA-4 are type I glycoproteins whose molecularweights are 44 kD and 41 to 43 kD, respectively. Both have animmunoglobulin-like domain, belong to the immunoglobulin superfamily,and have both function as a cell adhesion molecule and function as asignal transmission molecule.

[0010] Human CD28 forms a homodimer with a disulfide bond while CTLA-4exists as a monomer. Both CD28 and CTLA-4 genes are located at “2q33” onhuman chromosome and “1C” on mouse chromosome, and are composed of four(4) exons. Human CD28 and CTLA-4 are composed of 220 and 223 aminoacids, respectively, including the leader sequences, and amino acidhomology between them is 20 to 30%.

[0011] The ligands for CD28 and CTLA-4 are CD80 (B7-1) and CD86 (B7-2)in human and mice. CTLA-4 has about 20 times as high affinity to bothligands as CD28. It has been elucidated that the amino acid sequencestructures “MYPPPY (Met-Tyr-Pro-Pro-Pro-Tyr)” conserved through animalspecies is important for the binding of CD28 and CTLA-4 to CD80 (B7-1).It has also been reported that, when CD28 is stimulated, PI3 kinase(phosphoinositide 3 kinase, PI3K) associates with the phosphorylatedtyrosine residue in a partial sequence “YMNM (Tyr-Met-Asn-Met)” of CD28and that CD28 plays an important role in intracellular signaltransmission through this “YxxM” structure. Furthermore, it has beenreported that CTLA-4 also has a sequence represented by “YxxM,” namely“YVKM (Tyr-Val-Lys-Met)” in its cytoplasmic region and that, after beingstimulated, SYP associates with this sequence.

[0012] CD28 is expressed specifically in thymocytes and peripheral bloodT cells, and CTLA-4 is expressed specifically in activated T cells (CellEngineering: SUPPLEMENT, “Handbook of Adhesion Molecule”, Shujunsha,pp.93-102 (1994); ibid. pp.120-136; Experimental Medicine: SUPPLEMENT,“BIO SCIENCE Term Library, Immunity”, Yodosha, pp.94-98 (1995);Experimental Medicine: SUPPLEMENT, “BIO SCIENCE Term Library,Intracellular Signal Transduction”, Yodosha, pp.58-59 (1997); NihonRinsho, Vol.55, No.6, pp.215-220 (1997)).

[0013] In the regulation of T cell function (the activation and theinhibition of function of T cells), the importance of interactions amongmultiple molecules such as costimulator molecules (CD28, CD80 (B7-1),CD86 (B7-2), etc.) and CTLA-4, which cooperates with them, has thus beenrecognized, and this has been drawn attention to the relationshipbetween these molecules and diseases, and the treatment of diseases byregulating the function of these molecules.

[0014] As described above, although a living body activates its acquiredimmune response system against antigens that are foreign bodies to theliving body (self), it also has immunological tolerance so as to show noimmune response against its own component (autoantigen). Ifimmunological tolerance breaks down by some reason, immune response tothe autoantigen occurs, autoantigen-reactive T cells are induced by thesame mechanism as mentioned above to fall into abnormal state ofimmunity, and various autoimmune diseases are caused.

[0015] In other words, since non-stimulated antigen presenting cells(APC) in normal tissues do not express costimulatory molecules when theimmune system of a living body is normal, T cells are in theunresponsiveness state to maintain immunological tolerance even ifautoantigen-reactive T cells, which reacts with autoantigen, exist. Ithas been suggested that in abnormal state of immunity, moreautoantigen-reactive T cells are activated due to abnormal excess andcontinuous expression of costimulatory molecules to thereby causeautoimmune diseases.

[0016] From such viewpoints recently, many attempts to treat variousautoimmune diseases by modulating the transmission of costimulatorysignals, for example, the above-mentioned signal transmission betweenCD28/CTLA-4 and CD80/CD86, are proposed.

[0017] The results of such attempts have not yet clarified in detail themechanism of the T cell activation by interaction between costimulatorymolecules and the related molecules. Other unknown molecules may beinvolved in this mechanism.

[0018] Recently, there has been identified a novel co-stimulatorymolecule like the above-described “CD28” and “CTLA-4”, which is thoughtto carry out the transduction of a second signal (co-stimulatory signal)essential for the activation of lymphocytes such as T cells, andfunctional regulation coupled with said signal of activated lymphocytessuch as activated T cells. This molecule has been designated as AILIM(activation inducible lymphocyte immunomodulatory molecule) (in humans,mice and rats: Int. Immunol., Vol. 12, No. 1, p.51-55, 2000), alsoreferred to as ICOS (inducible co-stimulator) (in humans: Nature, Vol.397, No.6716, p.263-266, 1999)).

[0019] On the other hand, novel molecules celled B7h, B7RP-1, GL50 orLICOS which are ligands (AILIM ligands) interacting with thiscostimulatory transmission molecule AILIM (ICOS) have been identifiedvery recently (Nature. Vol.402, No.6763, pp.827-832, 1999; NatureMedicine, Vol.5, No.12, pp.1365-1369, 1999; J. Immunology, Vol.164,pp.1653-1657, 2000; Curr. Biol., Vol.10, No.6, pp.333-336, 2000).

[0020] The identification of these two kinds of novel molecules, namelyAILIM (ICOS) and B7RP-1 (B7h, GL50, LICOS), as the signal transductionpathway for the costimulatory signal essential for the above activationof lymphocytes such as T cells, and the control of the function ofactivated T cells, revealed that there is the novel third pathway by theinteraction between AILIM (ICOS) and B7RP-1 (B7h, GL50, LICOS), besidesthe known first and second signal pathways which are already knowntransduction pathway between CD28 and CD80 (B7-1)/CD86 (B7-2), and thatbetween CTLA4 and CD80 (B7-1)/CD 86 (B7-2).

[0021] Studies on the biological functions of these novel molecules, thefunction control of lymphocytes, such as T cells, through this thirdcostimulatory signal transduction by the molecules, and the relationshipbetween the novel signal transduction and diseases are in progress (J.Immunol., 166(1), pp.1, 2001; J. Immunol., 165(9), pp.5035, 2000;Biochem. Biophys. Res. Commun., 276(1), pp.335, 2000; Immunity, 13(1),pp.95, 2000; J. Exp. Med., 192(1), pp.53, 2000; Eur. J. Immunol., 30(4),pp.1040, 2000; WO 01/15732).

SUMMARY OF THE INVENTION

[0022] Specifically, an objective of the present invention is to revealbiological functions of the novel molecule AILIM, considered, like“CD28” and “CTLA-4”, as a molecule which transmits the secondary signal(costimulatory signal) essential for the activation of lymphocytes, suchas T cells, and which controls the functions of activated lymphocytes,such as activated T cells, by working with the signal; to revealrelationships between the expression of AILIM and diseases; and toprovide a method and a pharmaceutical which inhibit the development ofthe various diseases dependent on the expression pattern of AILIM orwhich treat the diseases by controlling the biological functions of theAILIM using the medical and pharmaceutical methods (for example, a drugsuch as a monoclonal antibody and a low molecular compound).

[0023] To achieve the above-described purposes, the present inventorshave actively pursued studies on human antibodies (particularly humanmonoclonal antibodies) against mammalian AILIMs (particularly humanAILIM), and as a result, by immunizing transgenic mice prepared usinggenetic recombination techniques so as to produce human antibodies withAILIM (specifically cell membrane fraction of cells expressing humanAILIM), succeeded first in the world in preparing a variety ofmonoclonal antibodies which bind to human AILIM, particularly thosewhich bind to human AILIM that regulate signal transduction mediated byhuman AILIM.

[0024] Since antibodies (particularly monoclonal antibodies) of thisinvention are derived from humans, they do not induce any severe immunerejection due to the immunogenicity against humans , HAMA (humananti-mouse antigenicity), in the host at all, which has been a bigproblem (side effect) in therapy using antibody pharmaceuticalscomprising antibodies derived from non-human mammals such as mice, andthus dramatically enhancing the value of antibody as medicine.

[0025] Therefore, human antibodies (particularly human monoclonalantibodies) which bind to mammalian AILIMs (particularly human AILIM) ofthis invention and pharmaceutical compositions comprising said humanantibodies (particularly human monoclonal antibodies) are useful asdrugs to control, with no induction of immune rejection due to HAMA inthe host, various physiological reactions related to the transduction ofco-stimulatory signal to AILIM-expressing cells mediated by AILIM (forexample, proliferation of AILIM-expressing cells, cytokine production byAILIM-expressing cells, immune cytolysis or apoptosis ofAILIM-expressing cells, and activity to induce antibody-dependentcytotoxicity to AILIM-expressing cells, and so on), and/or are alsouseful as drugs to suppress and prevent development of symptoms and/orprogress of various disorders related to the signal transductionmediated by said AILIM, and as medicine to treat or prevent saiddisorder.

[0026] Specifically, pharmaceutical compositions according to thisinvention are able to control (suppress or stimulate) proliferation ofAILIM-expressing cells or production of cytokine (for example,interferon g or interleukin 4, etc.) by AILIM-expressing cells, therebyenabling suppression of various pathological conditions and treatment orprevention of various disorders caused by diverse physiologicalphenomena related to signal transduction mediated by AILIM.

[0027] Use of pharmaceutical compositions according to this inventionenables suppression, prevention and/or treatment of, for example,various disorders (for example, rheumatoid arthritis, multiplesclerosis, autoimmune thyroiditis, allergic contact-type dermatitis,chronic inflammatory dermatosis such as lichen planus, systemic lupuserythematosus, insulin-dependent diabetes mellitus, psoriasis, etc.)classified into autoimmune or allergic disorders (particularlyautoimmune disease and delayed allergy caused by cellular immunity);arthropathia (for example, rheumatoid arthritis (RA) and osteoarthritis(OA)), inflammation (e.g., hepatitis); graft versus host reaction (GVHreaction); graft versus host disease (GVHD); immune rejectionaccompanying transplantation (homoplasty or heteroplasty) of a tissue(tissues such as skin, cornea, bone, etc.) or organ (liver, heart, lung,kidney, pancreas, etc.); immune response triggered by a foreign antigenor autoantigen (for example, production of antibodies against saidantigen, cell proliferation, production of cytokines); and disorderspossibly caused by the abnormal intestinal immunity (specificallyinflammatory intestinal disorders (particularly clone disease andulcerative colitis) and alimentary allergy).

[0028] Furthermore, in the field of suppression/treatment of immunerejection accompanying transplantation of above-described tissues andorgans, it is possible to augment the suppressive effect on transplantrejection of known immunosuppressant by using the pharmaceuticalcomposition of this invention together with said drugs which have beenutilized for suppression of immune rejection in such a transplantationtreatment.

[0029] Moreover, the pharmaceutical composition of the present inventioncan be applied for treating or preventing, any inflammatory diseases towhich various steroids are indicated as antiphlogistic.

[0030] The pharmaceutical composition of the present invention can beapplied to inflammatory disease for example, inflammation accompanyingvarious arthritis (for example, rheumatoid arthritis, osteoarthritis),pneumonia, hepatitis (including viral hepatitis), inflammationaccompanying infectious diseases, inflammatory bowel diseases,intestinal enteritis, nephritis (inflammation accompanying glomerularnephritis, nephrofibrosis), gastritis, angiitis, pancreatitis,peritonitis, bronchitis, myocarditis, cerebritis, inflammation inpostischemic reperfusion injury (myocardial ischemic reperfusioninjury), inflammation attributed to immune rejection aftertransplantation of tissue and organ, burn, various skin inflammation(psoriasis, allergic contact-type dermatitis, lichen planus which ischronic inflammatory skin disease), inflammation in multiple organfailure, inflammation after operation of PTCA or PTCR, and inflammationaccompanying arteriosclerosis, and autoimmune thyroiditis.

[0031] In addition, by using a method for identifying substances thatbind to AILIM or AILIM ligand, which is one of the present inventions,it becomes possible to screen to select pharmaceuticals (chemicalsynthetic compounds or antibodies) with potential activity to treatvarious disorders by binding to AILIM or AILIM ligands to regulatesignal transduction mediated by interaction of them.

[0032] Specifically, the present invention is the invention describedfrom the following (1) to (108).

[0033] (1) A human antibody which binds to AILIM.

[0034] (2) The human antibody of (1), wherein said AILIM is derived fromhuman.

[0035] (3) A human monoclonal antibody which binds to AILIM or a portionthereof.

[0036] (4) The human monoclonal antibody or a portion thereof of (3),wherein said AILIM is derived from human.

[0037] (5) The human monoclonal antibody or a portion thereof of (3) or(4), wherein said human monoclonal antibody has an activity to inhibit asignal transduction into a cell mediated by AILIM.

[0038] (6) The human monoclonal antibody or a portion thereof of (5),wherein said activity to inhibit a signal transduction is (a) or (b) ofthe followings:

[0039] (a) activity to inhibit proliferation of AILIM-expressing cells,or

[0040] (b) activity to inhibit cytokine production from AILIM-expressingcells.

[0041] (7) The human monoclonal antibody or a portion thereof of (6),wherein said cytokine is one of the cytokines produced by Th1-type orTh2-type T cell.

[0042] (8) The human monoclonal antibody or a portion thereof of (7),wherein said cytokine is interferon γ or interleukin 4.

[0043] (9) The human monoclonal antibody or a portion thereof of (5),wherein said human monoclonal antibody has an activity to prevent mixedlymphocyte reaction.

[0044] (10) The human monoclonal antibody or a portion thereof of (3) or(4), wherein said human monoclonal antibody has an activity to inducesignal transduction into a cell mediated by AILIM.

[0045] (11) The human monoclonal antibody or a portion thereof of (10),wherein said activity to induce signal transduction is (a) or (b) of thefollowings:

[0046] (a) activity to induce proliferation of AILIM-expressing cells,or

[0047] (b) activity to induce cytokine production from AILIM-expressingcells.

[0048] (12) The human monoclonal antibody or a portion thereof of (11),wherein said cytokine is one of the cytokines produced by Th1-type orTh2-type T cell.

[0049] (13) The human monoclonal antibody or a portion thereof of (12),wherein said cytokine is interferon γ or interleukin 4.

[0050] (14) The human monoclonal antibody or a portion thereof of (3) or(4), wherein said human monoclonal antibody has an activity to induceantibody-dependent cytotoxicity to AILIM-expressing cells, and/or immunecytolysis or apoptosis of AILIM-expressing cells.

[0051] (15) The human monoclonal antibody or a portion thereof of (3) or(4), wherein the binding rate constant (ka) between said monoclonalantibody and AILIM is 1.0×10³ (1/M.Sec) or more.

[0052] (16) The human monoclonal antibody or a portion thereof of (15),wherein said binding rate constant (ka) is 1.0×10⁴ (1/M.Sec) or more.

[0053] (17) The human monoclonal antibody or a portion thereof of (16),wherein said binding rate constant (ka) is 1.0×10⁵ (1/M.Sec) or more.

[0054] (18) The human monoclonal antibody or a portion thereof of (3) or(4), wherein the dissociation rate constant (kd) between said monoclonalantibody and AILIM is 1.0×10⁻³ (1/Sec) or less.

[0055] (19) The human monoclonal antibody or a portion thereof of (18),wherein said dissociation rate constant (kd) is 1.0×10⁻⁴ (1/Sec) orless.

[0056] (20) The human monoclonal antibody or a portion thereof of (19),wherein said dissociation rate constant (kd) is 1.0×10⁻⁵ (1/Sec) orless.

[0057] (21) The human monoclonal antibody or a portion thereof of (3) or(4), wherein the dissociation constant (Kd) between said monoclonalantibody and AILIM is 1.0×10⁻⁶ (M) or less.

[0058] (22) The human monoclonal antibody or a portion thereof of (21),wherein said dissociation constant (Kd) is 1.0×10⁻⁷ (M) or less.

[0059] (23) The human monoclonal antibody or a portion thereof of (22),wherein said dissociation constant (Kd) is 1.0×10⁻⁸ (M) or less.

[0060] (24) The human monoclonal antibody or a portion thereof of (23),wherein said dissociation constant (Kd) is 1.0×10⁻⁹ (M) or less.

[0061] (25) The human monoclonal antibody or a portion thereof of (4),wherein a V region DNA encoding a heavy chain variable region of saidhuman monoclonal antibody is derived from either the humanimmunoglobulin heavy chain V gene segment 1-02 or 3-13.

[0062] (26) The human monoclonal antibody or a portion thereof of (4),wherein a V region DNA encoding a light chain variable region of saidhuman monoclonal antibody is derived from either the humanimmunoglobulin light chain V gene segment L5 or A27.

[0063] (27) The human monoclonal antibody or a portion thereof of (25)or (26), wherein a V region DNA encoding a heavy chain variable regionof said human monoclonal antibody is derived from either the humanimmunoglobulin heavy chain V gene segment 1-02 or 3-13, and wherein a Vregion DNA encoding a light chain variable region of said humanmonoclonal antibody is derived from either the human immunoglobulinlight chain V gene segment L5 or A27.

[0064] (28) The human monoclonal antibody or a portion thereof of (27),wherein the V region DNA encoding a heavy chain variable region of saidhuman monoclonal antibody is derived from the human immunoglobulin heavychain V gene segment 1-02, and the V region DNA encoding a light chainvariable region of said human monoclonal antibody is derived from thehuman immunoglobulin light chain V gene segment L5.

[0065] (29) The human monoclonal antibody or a portion thereof of (27),wherein the V region DNA encoding a heavy chain variable region of saidhuman monoclonal antibody is derived from the human immunoglobulin heavychain V gene segment 3-13, and the V region DNA encoding a light chainvariable region of said human monoclonal antibody is derived from thehuman immunoglobulin light chain V gene segment A27.

[0066] (30) The human monoclonal antibody or a portion thereof of (4),wherein a heavy chain variable region of said human monoclonal antibodyhas an amino acid sequence defined in any of the following (a) through(f):

[0067] (a) amino acid sequence comprising amino acids from position 20through 117 of SEQ ID NO:28,

[0068] (b) amino acid sequence comprising amino acids from position 20through 117 of SEQ ID NO:28 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0069] (c) amino acid sequence comprising amino acids from position 20through 116 of SEQ ID NO:32,

[0070] (d) amino acid sequence comprising amino acids from position 20through 116 of SEQ ID NO:32 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0071] (e) amino acid sequence comprising amino acids from position 20through 116 of SEQ ID NO:36, or

[0072] (f) amino acid sequence comprising amino acids from position 20through 116 of SEQ ID NO:36, in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added.

[0073] (31) The human monoclonal antibody or a portion thereof of (4),wherein a heavy chain polypeptide of said human monoclonal antibody hasan amino acid sequence defined in any of the following (a) through (f):

[0074] (a) amino acid sequence comprising amino acids from position 20through 470 of SEQ ID NO:28,

[0075] (b) amino acid sequence comprising amino acids from position 20through 470 of SEQ ID NO:28 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0076] (c) amino acid sequence comprising amino acids from position 20through 470 of SEQ ID NO:32,

[0077] (d) amino acid sequence comprising amino acids from position 20through 470 of SEQ ID NO:32 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0078] (e) amino acid sequence comprising amino acids from position 20through 470 of SEQ ID NO:36, or

[0079] (f) amino acid sequence comprising amino acids from position 20through 470 of SEQ ID NO:36 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0080] (32) The human monoclonal antibody or a portion thereof of (4),wherein a light chain variable region of said human monoclonal antibodyhas an amino acid sequence defined in any of the following (a) through(f):

[0081] (a) amino acid sequence comprising amino acids from position 23through 116 of SEQ ID NO:30,

[0082] (b) amino acid sequence comprising amino acids from position 23through 116 of SEQ ID NO:30 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0083] (c) amino acid sequence comprising amino acids from position 21through 116 of SEQ ID NO:34,

[0084] (d) amino acid sequence comprising amino acids from position 21through 116 of SEQ ID NO:34 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0085] (e) amino acid sequence comprising amino acids from position 21through 116 of SEQ ID NO:38, or

[0086] (f) amino acid sequence comprising amino acids from position 21through 116 of SEQ ID NO:38 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0087] (33) The human monoclonal antibody or a portion thereof of (4),wherein a light chain polypeptide of said human monoclonal antibody hasan amino acid sequence defined in any of the following (a) through (f):

[0088] (a) amino acid sequence comprising amino acids from position 23through 236 of SEQ ID NO:30,

[0089] (b) amino acid sequence comprising amino acids from position 23through 236 of SEQ ID NO:30 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0090] (c) amino acid sequence comprising amino acids from position 21through 236 of SEQ ID NO:34,

[0091] (d) amino acid sequence comprising amino acids from position 21through 236 of SEQ ID NO:34 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0092] (e) amino acid sequence comprising amino acids from position 21through 236 of SEQ ID NO:38, or

[0093] (f) amino acid sequence comprising amino acids from position 21through 236 of SEQ ID NO:38 in which one or more amino acid residues aredeleted or substituted, or to which one or more amino acid residues areinserted or added.

[0094] (34) The human monoclonal antibody or a portion thereof of (4),wherein said human monoclonal antibody has the following characteristics(a) and (b):

[0095] (a) a heavy chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 117according to SEQ ID NO:28, and

[0096] (b) a light chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 23 through 116according to SEQ ID NO:30.

[0097] (35) The human monoclonal antibody or a portion thereof of (4),wherein said human monoclonal antibody has the following characteristics(a) and (b):

[0098] (a) a heavy chain polypeptide has an amino acid sequence fromamino acid 20 through 470 according to SEQ ID NO:28, and

[0099] (b) a light chain polypeptide has an amino acid sequence fromamino acid 23 through 236 according to SEQ ID NO:30.

[0100] (36) The human monoclonal antibody or a portion thereof of (4),wherein said human monoclonal antibody has the following characteristics(a) and (b):

[0101] (a) a heavy chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 116according to SEQ ID NO:32, and

[0102] (b) a light chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 21 through 116according to SEQ ID NO:34.

[0103] (37) The human monoclonal antibody or a portion thereof of (4),wherein said human monoclonal antibody has the following characteristics(a) and (b):

[0104] (a) a heavy chain polypeptide has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 470according to SEQ ID NO:32, and

[0105] (b) a light chain polypeptide has an amino acid sequencecomprising the amino acid sequence from amino acid 21 through 236according to SEQ ID NO:34.

[0106] (38) The human monoclonal antibody or a portion thereof of (4),wherein said human monoclonal antibody has the following characteristics(a) and (b):

[0107] (a) a heavy chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 116according to SEQ ID NO:36, and

[0108] (b) a light chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 21 through 116according to SEQ ID NO:38.

[0109] (39) The human monoclonal antibody or a portion thereof of (4),wherein said human monoclonal antibody has the following characteristics(a) and (b):

[0110] (a) a heavy chain polypeptide has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 470according to SEQ ID NO:36, and

[0111] (b) a light chain polypeptide has an amino acid sequencecomprising the amino acid sequence from amino acid 21 through 236according to SEQ ID NO:38.

[0112] (40) The human monoclonal antibody or a portion thereof of anyone of (3) through (29), wherein said human monoclonal antibody is amonoclonal antibody derived from a transgenic non-human mammal capableof producing human antibodies.

[0113] (41) The human monoclonal antibody or a portion thereof of (40),wherein said human monoclonal antibody is obtained by immunizingtransgenic non-human mammal capable of producing human antibody withAILIM-expressing cells, membrane fractions derived from said cells,whole molecules constituting AILIM or a portion thereof, or genesencoding AILIM or a portion thereof.

[0114] (42) The human monoclonal antibody or a portion thereof of (40)or (41), wherein said transgenic non-human mammal is a transgenic mouse.

[0115] (43) A DNA or a portion thereof encoding a polypeptide selectedfrom the group consisting of (a) through (f) below:

[0116] (a) a polypeptide comprising the amino acid sequence from aminoacid 20 through 117 according to SEQ ID NO:28,

[0117] (b) a polypeptide comprising the amino acid sequence from aminoacid 23 through 116 according to SEQ ID NO:30,

[0118] (c) a polypeptide comprising the amino acid sequence from aminoacid 20 through 116 according to SEQ ID NO:32,

[0119] (d) a polypeptide comprising the amino acid sequence from aminoacid 21 through 116 according to SEQ ID NO:34,

[0120] (e) a polypeptide comprising the amino acid sequence from aminoacid 20 through 116 according to SEQ ID NO:36, and

[0121] (f) a polypeptide comprising the amino acid sequence from aminoacid 21 through 116 according to SEQ ID NO:38.

[0122] (44) A DNA or a portion thereof encoding a polypeptide selectedfrom the group consisting of (a) through (f) below:

[0123] (a) a polypeptide comprising the amino acid sequence from aminoacids 20 through 470 according to SEQ ID NO:28,

[0124] (b) a polypeptide comprising the amino acid sequence from aminoacids 23 through 236 according to SEQ ID NO:30,

[0125] (c) a polypeptide comprising the amino acid sequence from aminoacids 20 through 470 according to SEQ ID NO:32,

[0126] (d) a polypeptide comprising the amino acid sequence from aminoacids 21 through 236 according to SEQ ID NO:34,

[0127] (e) a polypeptide comprising the amino acid sequence from aminoacids 20 through 470 according to SEQ ID NO:36, and

[0128] (f) a polypeptide comprising the amino acid sequence from aminoacids 21 through 236 according to SEQ ID NO:38.

[0129] (45) A DNA or a portion thereof selected from the groupconsisting of (a) through (f) below:

[0130] (a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27,

[0131] (b) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29,

[0132] (c) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31,

[0133] (d) a DNA comprising the nucleotide sequence from nucleotides 88through 375 according to SEQ ID NO:33,

[0134] (e) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35, and

[0135] (f) a DNA comprising the nucleotide sequence from nucleotides 93through 380 according to SEQ ID NO:37.

[0136] (46) A DNA or a portion thereof selected from a group consistingof (a) through (f) below:

[0137] (a) a DNA comprising the nucleotide sequence from nucleotides 69through 1481 according to SEQ ID NO:27,

[0138] (b) a DNA comprising the nucleotide sequence from nucleotides 39through 749 according to SEQ ID NO:29,

[0139] (c) a DNA comprising the nucleotide sequence from nucleotides 94through 1506 defined in SEQ ID NO:31,

[0140] (d) a DNA comprising the nucleotide sequence from nucleotides 28through 738 according to SEQ ID NO:33,

[0141] (e) a DNA comprising the nucleotide sequence from nucleotides 96through 1508 according to SEQ ID NO:35, and

[0142] (f) a DNA comprising the nucleotide sequence from nucleotides 33through 743 according to SEQ ID NO:37.

[0143] (47) A vector comprising the DNA of any one of (43) through (46).

[0144] (48) The vector of (47) comprising a DNA according to any of thefollowing (a) through (c):

[0145] (a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27,

[0146] (b) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, or

[0147] (c) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35.

[0148] (49) The vector of (47) comprising a DNA according to any of thefollowing (a) through (c):

[0149] (a) a DNA comprising the nucleotide sequence from nucleotides 69through 1481 according to SEQ ID NO:27,

[0150] (b) a DNA comprising the nucleotide sequence from nucleotides 94through 1506 according to SEQ ID NO:31, or

[0151] (c) a DNA comprising the nucleotide sequence from nucleotides 96through 1508 according to SEQ ID NO:35.

[0152] (50) The vector of (47) comprising a DNA according to any of thefollowing (a) through (c):

[0153] (a) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29,

[0154] (b) a DNA comprising the nucleotide sequence from nucleotides 88through 375 according to SEQ ID NO:33, or

[0155] (c) a DNA comprising the nucleotide sequence from nucleotides 93through 380 according to SEQ ID NO:37.

[0156] (51) The vector of (47) comprising a DNA according to any of thefollowing (a) through (c):

[0157] (a) a DNA comprising the nucleotide sequence from nucleotides 39through 749 according to SEQ ID NO:29,

[0158] (b) a DNA comprising the nucleotide sequence from nucleotides 28through 738 according to SEQ ID NO:33, or

[0159] (c) a DNA comprising the nucleotide sequence from nucleotides 33through 743 according to SEQ ID NO:37.

[0160] (52) The vector of (47) comprising a DNA according to thefollowing (a) and (b):

[0161] (a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27, and

[0162] (b) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29.

[0163] (53) The vector of (47) comprising a DNA according to thefollowing (a) and (b):

[0164] (a) a DNA comprising the nucleotide sequence from nucleotides 69through 1481 according to SEQ ID NO:27, and

[0165] (b) a DNA comprising the nucleotide sequence from nucleotides 39through 749 according to SEQ ID NO:29.

[0166] (54) The vector of (47) comprising a DNA according to thefollowing (a) and (b):

[0167] (a) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, and

[0168] (b) a DNA comprising the nucleotide sequence from nucleotides 88through 375 according to SEQ ID NO:33.

[0169] (55) The vector of (47) comprising a DNA according to thefollowing (a) and (b):

[0170] (a) a DNA comprising the nucleotide sequence from nucleotides 94through 1506 according to SEQ ID NO:31, and

[0171] (b) a DNA comprising the nucleotide sequence from nucleotides 28through 738 according to SEQ ID NO:33.

[0172] (56) The vector of (47) comprising a DNA according to thefollowing (a) and (b):

[0173] (a) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35, and

[0174] (b) a DNA comprising the nucleotide sequence from nucleotides 93through 380 according to SEQ ID NO:37.

[0175] (57) The vector of (47) comprising a DNA according to thefollowing (a) and (b):

[0176] (a) a DNA comprising the nucleotide sequence from nucleotides 96through 1508 according to SEQ ID NO:35, and

[0177] (b) a DNA comprising the nucleotide sequence from nucleotides 33through 743 according to SEQ ID NO:37.

[0178] (58) A cell producing a human monoclonal antibody of any one of(3) through (42).

[0179] (59) The cell of (58), wherein said cell is a fused cell obtainedby fusing B cell, derived from a mammal capable of producing said humanmonoclonal antibody, and myeloma cell derived from a mammal.

[0180] (60) A genetic recombinant host transformed by transferring a DNAdescribed below in (a) or a vector comprising said DNA, a DNA describedbelow in (b) or a vector comprising said DNA, or both DNAs describedbelow in (a) and (b) or a vector comprising both of said DNAs:

[0181] (a) a DNA encoding a heavy chain polypeptide or a portion thereofof a monoclonal antibody which binds to human AILIM; or

[0182] (b) a DNA encoding a light chain polypeptide or a portion thereofof a monoclonal antibody which binds to human AILIM.

[0183] (61) The genetic recombinant host of (60), wherein saidmonoclonal antibody is a human monoclonal antibody.

[0184] (62) The genetic recombinant host of (60) or (61), wherein saidhost is a mammalian cell.

[0185] (63) The genetic recombinant host of (60) or (61), wherein saidhost is a mammalian fertilized egg.

[0186] (64) The genetic recombinant host of any one of (60) through(63), wherein said heavy chain polypeptide is one of the heavy chainpolypeptides selected from the group consisting of the following (a)through (c):

[0187] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 117 according to SEQ ID NO:28,

[0188] (b) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 116 according to SEQ ID NO:32, and

[0189] (c) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 116 according to SEQ ID NO:36.

[0190] (65) The genetic recombinant host of any one of (60) through(63), wherein said heavy chain polypeptide is one of the heavy chainpolypeptide selected from the group consisting of the following (a)through (c):

[0191] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 470 according to SEQ ID NO:28,

[0192] (b) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 470 according to SEQ ID NO:32, and

[0193] (c) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 470 according to SEQ ID NO:36.

[0194] (66) The genetic recombinant host of any one of (60) through(63), wherein said light chain polypeptide is one of the light chainpolypeptide selected from the group consisting of the following (a)through (c):

[0195] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 23 through 116 according to SEQ ID NO:30,

[0196] (b) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 21 through 116 according to SEQ ID NO:34, and

[0197] (c) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 21 through 116 according to SEQ ID NO:38.

[0198] (67) The genetic recombinant host of any one of (60) through(63), wherein said light chain polypeptide is one of the light chainpolypeptide selected from the group consisting of the following (a)through (c):

[0199] (a) a light chain polypeptide comprising the amino acid sequencefrom amino acids 23 through 236 according to SEQ ID NO:30,

[0200] (b) a light chain polypeptide comprising the amino acid sequencefrom amino acids 21 through 236 according to SEQ ID NO:34, and

[0201] (c) a light chain polypeptide comprising the amino acid sequencefrom amino acids 21 through 236 according to SEQ ID NO:38.

[0202] (68) The genetic recombinant host of any one of (60) through(63), wherein said heavy chain and light chain polypeptides are thosedefined below in (a) and (b), respectively:

[0203] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 117 according to SEQ ID NO:28, and

[0204] (b) a light chain polypeptide comprising the amino acid sequencefrom amino acids 23 through 116 according to SEQ ID NO:30.

[0205] (69) The genetic recombinant host of any one of (60) through(63), wherein said heavy chain and light chain polypeptides are thosedefined below in (a) and (b), respectively:

[0206] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 470 according to SEQ ID NO:28, and

[0207] (b) a light chain polypeptide comprising the amino acid sequencefrom amino acids 23 through 236 according to SEQ ID NO:30.

[0208] (70) The genetic recombinant host of any one of (60) through(63), wherein said heavy chain and light chain polypeptides are thosedefined below in (a) and (b), respectively:

[0209] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 116 according to SEQ ID NO: 32, and

[0210] (b) a light chain polypeptide comprising the amino acid sequencefrom amino acids 21 through 116 according to SEQ ID NO:34.

[0211] (71) The genetic recombinant host of any one of (60) through(63), wherein said heavy chain and light chain polypeptides are thosedefined below in (a) and (b), respectively:

[0212] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 470 according to SEQ ID NO:32, and

[0213] (b) a light chain polypeptide comprising the amino acid sequencefrom amino acids 21 through 236 according to SEQ ID NO:34.

[0214] (72) The genetic recombinant host of any one of (60) through(63), wherein said heavy chain and light chain polypeptides are thosedefined below in (a) and (b), respectively:

[0215] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 116 according to SEQ ID NO:36, and

[0216] (b) a light chain polypeptide comprising the amino acid sequencefrom amino acids 21 through 116 according to SEQ ID NO:38.

[0217] (73) The genetic recombinant host of any one of (60) through(63), wherein said heavy chain and light chain polypeptides are thosedefined below in (a) and (b), respectively:

[0218] (a) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 470 according to SEQ ID NO:36, and

[0219] (b) a light chain polypeptide comprising the amino acid sequencefrom amino acids 21 through 236 according to SEQ ID NO:38.

[0220] (74) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said heavy chain polypeptide is a DNAdefined in any of following (a) through (c):

[0221] (a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27,

[0222] (b) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, and

[0223] (c) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35.

[0224] (75) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said heavy chain polypeptide is a DNAdefined in any of following (a) through (c):

[0225] (a) a DNA comprising the nucleotide sequence from nucleotides 69through 1481 according to SEQ ID NO:27,

[0226] (b) a DNA comprising the nucleotide sequence from nucleotides 94through 1506 according to SEQ ID NO:31, and

[0227] (c) a DNA comprising the nucleotide sequence from nucleotides 96through 1508 according to SEQ ID NO:35.

[0228] (76) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said light chain polypeptide is a DNAdefined in any of following (a) through (c):

[0229] (a) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29,

[0230] (b) a DNA comprising the nucleotide sequence from nucleotides 88through 375 according to SEQ ID NO:33, and

[0231] (c) a DNA comprising the nucleotide sequence from nucleotides 93through 380 according to SEQ ID NO:37.

[0232] (77) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said light chain polypeptide is a DNA asdefined in any of following (a) through (c):

[0233] (a) a DNA comprising the nucleotide sequence from nucleotides 39through 749 according to SEQ ID NO:29,

[0234] (b) a DNA comprising the nucleotide sequence from nucleotides 28through 738 according to SEQ ID NO:33, and

[0235] (c) a DNA comprising the nucleotide sequence from nucleotides 33through 743 according to SEQ ID NO:37.

[0236] (78) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said heavy chain polypeptide is a DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is a DNA as described below in (b):

[0237] (a) a DNA comprising the nucleotide sequence from nucleotides 126through 419 according to SEQ ID NO:27, and

[0238] (b) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29.

[0239] (79) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

[0240] (a) a DNA comprising the nucleotide sequence from nucleotides 69through 1481 according to SEQ ID NO:27, and

[0241] (b) a DNA comprising the nucleotide sequence from nucleotides 39through 749 according to SEQ ID NO:29.

[0242] (80) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

[0243] (a) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, and

[0244] (b) a DNA comprising the nucleotide sequence from nucleotides 88through 375 V SEQ ID NO:33.

[0245] (81) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

[0246] (a) a DNA comprising the nucleotide sequence from nucleotides 94through 1506 according to SEQ ID NO:31, and

[0247] (b) a DNA comprising the nucleotide sequence from nucleotides 28through 738 according to SEQ ID NO:33.

[0248] (82) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

[0249] (a) a DNA comprising the nucleotide sequence from nucleotides 153through 443 according to SEQ ID NO:35, and

[0250] (b) a DNA comprising the nucleotide sequence from nucleotides 93through 380 according to SEQ ID NO:37.

[0251] (83) The genetic recombinant host of any one of (60) through(63), wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b):

[0252] (a) a DNA comprising the nucleotide sequence from nucleotides 96through 1508 according to SEQ ID NO:35, and

[0253] (b) a DNA comprising the nucleotide sequence from nucleotides 33through 743 according to SEQ ID NO:37.

[0254] (84) A human monoclonal antibody or a portion thereof produced bya genetic recombinant host (provided excluding the case where said hostis a fertilized egg) of any one of (60) through (62), or of any one of(64) through (83).

[0255] (85) A pharmaceutical composition comprising the human antibodyof (1) or (2), and a pharmaceutically acceptable carrier.

[0256] (86) A pharmaceutical composition comprising the human monoclonalantibody or a portion thereof of any one of (3) to (42), and apharmaceutically acceptable carrier.

[0257] (87) A pharmaceutical composition comprising a human monoclonalantibody or a portion thereof of (84), and a pharmaceutically acceptablecarrier.

[0258] (88) The pharmaceutical composition of any one of (85) through(87), wherein said pharmaceutical composition is used to inhibit signaltransduction into the cell mediated by AILIM.

[0259] (89) The pharmaceutical composition of any one of (85) through(87), wherein said pharmaceutical composition is used to preventproliferation of AILIM-expressing cells.

[0260] (90) The pharmaceutical composition of any one of (85) through(87), wherein said pharmaceutical composition is used to preventproduction of a cytokine from AILIM-expressing cells.

[0261] (91) The pharmaceutical composition of any one of (85) through(87), wherein said pharmaceutical composition is used to induce signaltransduction into a cell mediated by AILIM.

[0262] (92) The pharmaceutical composition of any one of (85) through(87), wherein said pharmaceutical composition is used to induceproliferation of AILIM-expressing cells.

[0263] (93) The pharmaceutical composition of any one of (85) through(87), wherein said pharmaceutical composition is used to induceproduction of a cytokine from AILIM-expressing cells.

[0264] (94) The pharmaceutical composition of any one of (85) through(87), wherein said pharmaceutical composition is used to induceantibody-dependent cytotoxicity against AILIM-expressing cells, and/orimmune cytolysis or apoptosis of AILIM-expressing cells.

[0265] (95) A pharmaceutical composition for preventing, treating, orprophylaxis of delayed type allergy, comprising a substance having anactivity in modulating signal transduction mediated by AILIM, and apharmaceutically acceptable carrier.

[0266] (96) The pharmaceutical composition of (95), wherein thesubstance is a protein substance.

[0267] (97) The pharmaceutical composition of (96), wherein the proteinsubstance is selected from the group consisting of:

[0268] a) an antibody which binds to AILIM or a portion thereof;

[0269] b) a polypeptide comprising the whole or a portion of anextracellular region of AILIM;

[0270] c) a fusion polypeptide comprising the whole or a portion of anextracellular region of AILIM, and the whole or a portion of a constantregion of immunoglobulin heavy chain; and

[0271] d) a polypeptide which binds to AILIM.

[0272] (98) The pharmaceutical composition of (97), wherein saidantibody that binds to AILIM is the human antibody of (1) or (2).

[0273] (99) The pharmaceutical composition of (97), wherein saidantibody that binds to AILIM is the human monoclonal antibody of any oneof (3) through (42).

[0274] (100) The pharmaceutical composition of (97), wherein saidantibody against AILIM is the human monoclonal antibody of (84).

[0275] (101) The pharmaceutical composition of (95), wherein thesubstance is a non-protein substance.

[0276] (102) The pharmaceutical composition of (101), wherein thenon-protein substance is DNA, RNA, or a chemically synthesized compound.

[0277] (103) A method for identifying substances that bind to AILIM orAILIM ligand comprising the following processes:

[0278] (a) preparing an insoluble carrier on which the entireextracellular region of AILIM or a portion thereof is immobilized;

[0279] (b) preparing a polypeptide comprising the whole extracellularregion of AILIM ligand or a portion thereof labeled with a labelingmaterial that emit a detectable signal;

[0280] (c) reacting the insoluble carrier in process(a) with thepolypeptide in process (b);

[0281] (d) reacting the insoluble carrier of process (a), thepolypeptide of process (b) and said substance to each other in anyarbitrary orders;

[0282] (e) detecting the signal emitted from said labeling materialcontained in the complex produced in process (c), and the signal emittedfrom said labeling material contained in the complex produced in process(d), respectively; and

[0283] (f) comparing the magnitude of each of signals detected inprocess (e).

[0284] (104) A method for identifying substances that bind to AILIM orAILIM ligand comprising the following processes:

[0285] (a) preparing an insoluble carrier on which the entireextracellular region of AILIM ligand or a portion thereof isimmobilized;

[0286] (b) preparing a polypeptide comprising the whole extracellularregion of AILIM or a portion thereof labeled with a labeling materialthat emit a detectable signal;

[0287] (c) reacting the insoluble carrier in process (a) with thepolypeptide in process (b);

[0288] (d) reacting the insoluble carrier of process (a), thepolypeptide of process (b) and said substance to each other in anyarbitrary orders;

[0289] (e) detecting the signal emitted from said labeling materialcontained in the complex produced in process (c), and the signal emittedfrom said labeling material contained in the complex produced in process(d), respectively; and

[0290] (f) comparing the magnitude of each of signals detected inprocess (e).

[0291] (105) The method of (103) or (104), wherein said polypeptidecomprising the whole extracellular region of AILIM or a portion thereofis a fusion polypeptide comprising a polypeptide, comprising the wholeextracellular region of AILIM or a portion thereof, and the wholeconstant region of immunoglobulin heavy chain or a portion thereof.

[0292] (106) The method of (103) or (104), wherein said polypeptidecomprising the whole extracellular region of AILIM ligand or a portionthereof is a fusion polypeptide comprising a polypeptide, comprising thewhole extracellular region of AILIM ligand or a portion thereof, and thewhole constant region of immunoglobulin heavy chain or a portionthereof.

[0293] (107) The method of any one of (103) through (106), wherein saidAILIM is a human AILIM.

[0294] (108) The method of any one of (103) through (107), wherein saidAILIM ligand is a human AILIM ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

[0295]FIG. 1 shows respective reactivities of anti-human IgG antibody,anti-human Igκ antibody and anti-human IgFc antibody to the humananti-human AILIM monoclonal antibody, analyzed by cell ELISA using aflow cytometer.

[0296] Panels (a) to (1) show respective results of the assays indicatedbelow.

[0297] Panel (a): result of assay in which biotin-labeled anti-human IgGantibody as a secondary antibody was added in the absence of primaryantibody into the microplate where wild-type HPB-ALL cells had beenplated.

[0298] Panel (b): result of assay in which biotin-labeled anti-human Igκantibody as a secondary antibody was added in the absence of primaryantibody into the microplate where wild-type HPB-ALL cells had beenplated.

[0299] Panel (c): result of assay in which biotin-labeled anti-humanIgFc antibody as a secondary antibody was added in the absence ofprimary antibody into the microplate where wild-type HPB-ALL cells hadbeen plated.

[0300] Panel (d): result of assay in which human anti-human AILIMmonoclonal antibody JMab-136 was used as a primary antibody andbiotin-labeled anti-human IgG antibody was used as a secondary antibody.

[0301] Panel (e): result of assay in which human anti-human AILIMmonoclonal antibody JMab-136 was used as a primary antibody andbiotin-labeled anti-human Igκ antibody was used as a secondary antibody.

[0302] Panel (f): result of assay in which human anti-human AILIMmonoclonal antibody JMab-136 was used as a primary antibody andbiotin-labeled anti-human IgFc antibody was used as a secondaryantibody.

[0303] Panel (g): result of assay in which human anti-human AILIMmonoclonal antibody JMab-138 was used as a primary antibody andbiotin-labeled anti-human IgG antibody was used as a secondary antibody.

[0304] Panel (h): result of assay in which human anti-human AILIMmonoclonal antibody JMab-138 was used as a primary antibody andbiotin-labeled anti-human Igκ antibody was used as a secondary antibody.

[0305] Panel (i): result of assay in which human anti-human AILIMmonoclonal antibody JMab-138 was used as a primary antibody andbiotin-labeled anti-human IgFc antibody was used as a secondaryantibody.

[0306] Panel (j): result of assay in which human anti-human AILIMmonoclonal antibody JMab-139 was used as a primary antibody andbiotin-labeled anti-human IgG antibody was used as a secondary antibody.

[0307] Panel (k): result of assay in which human anti-human AILIMmonoclonal antibody JMab-139 was used as a primary antibody andbiotin-labeled anti-human Igκ antibody was used as a secondary antibody.

[0308] Panel (l): result of assay in which human anti-human AILIMmonoclonal antibody JMab-139 was used as a primary antibody andbiotin-labeled anti-human IgFc antibody was used as a secondaryantibody.

[0309] The curve with open symbols in each panel corresponds to theresult of assay in which human anti-KLH monoclonal antibody was used asthe control antibody.

[0310]FIG. 2 shows a calibration curve with respect to human IgGmonoclonal antibody (standard material) assayed by sandwich ELISA usinganti-human IgG antibody.

[0311] The vertical axis indicates fluorescence intensity, and thehorizontal axis indicates the concentration of the standard material.

[0312]FIG. 3 shows binding activities of various mouse anti-human AILIMmonoclonal antibodies to human AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

[0313] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0314] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “human” indicates the result of abinding assay to the human AILIM-overexpressing recombinant CHO cell.

[0315]FIG. 4 shows binding activities of various human anti-human AILIMmonoclonal antibodies or human anti-KLH monoclonal antibodies as anegative control to human AILIM-overexpressing recombinant CHO cells orwild-type CHO cells.

[0316] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0317] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “human” indicates the result of abinding assay to the human AILIM-overexpressing recombinant CHO cell.

[0318]FIG. 5 shows binding activities of various human anti-human AILIMmonoclonal antibodies to human AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

[0319] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0320] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “human” indicates the result of abinding assay to the human AILIM-overexpressing recombinant CHO cell.

[0321]FIG. 6 shows binding activities of various human anti-human AILIMmonoclonal antibodies to human AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

[0322] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0323] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “human” indicates the result of abinding assay to the human AILIM-overexpressing recombinant CHO cell.

[0324]FIG. 7 shows binding activities of rat anti-human AILIM monoclonalantibodies to mouse AILIM-overexpressing recombinant CHO cells orwild-type CHO cells.

[0325] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0326] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “mouse” indicates the result of abinding assay to the mouse AILIM-overexpressing recombinant CHO cell.

[0327]FIG. 8 shows binding activities of various human anti-human AILIMmonoclonal antibodies or human anti-KLH monoclonal antibodies as anegative control to mouse AILIM-overexpressing recombinant CHO cells orwild-type CHO cells.

[0328] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0329] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “mouse” indicates the result of abinding assay to the mouse AILIM-overexpressing recombinant CHO cell.

[0330]FIG. 9 shows binding activities of various human anti-human AILIMmonoclonal antibodies to mouse AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

[0331] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0332] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “mouse” indicates the result of abinding assay to the mouse AILIM-overexpressing recombinant CHO cell.

[0333]FIG. 10 shows binding activities of various human anti-human AILIMmonoclonal antibodies to mouse AILIM-overexpressing recombinant CHOcells or wild-type CHO cells.

[0334] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0335] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “mouse” indicates the result ofbinding assay to the mouse AILIM-overexpressing recombinant CHO cell.

[0336]FIG. 11 shows binding activities of various mouse anti-rat AILIMmonoclonal antibodies to rat AILIM-overexpressing recombinant CHO cellsor wild-type CHO cells.

[0337] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0338] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “rat” indicates the result of abinding assay to the rat AILIM-overexpressing recombinant CHO cell.

[0339]FIG. 12 shows binding activities of various human anti-human AILIMmonoclonal antibodies or human anti-KLH monoclonal antibodies as anegative control to rat AILIM-overexpressing recombinant CHO cells orwild-type CHO cells.

[0340] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0341] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “rat” indicates the result of abinding assay to the rat AILIM-overexpressing recombinant CHO cell.

[0342]FIG. 13 shows binding activities of various human anti-human AILIMmonoclonal antibodies to rat AILIM-overexpressing recombinant CHO cellsor wild-type CHO cells.

[0343] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0344] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “rat” indicates the result of abinding assay to the rat AILIM-overexpressing recombinant CHO cell.

[0345]FIG. 14 shows binding activities of various human anti-human AILIMmonoclonal antibodies to rat AILIM-overexpressing recombinant CHO cellsor wild-type CHO cells.

[0346] The vertical axis indicates fluorescence intensity as an index ofbinding activity to the recombinant cells, and the horizontal axisindicates the concentration of the antibody added.

[0347] The term “CHO” in the figure indicates the result of a bindingassay to the wild-type CHO cell, and “rat” indicates the result of abinding assay to the rat AILIM-overexpressing recombinant CHO cell.

[0348]FIG. 15 shows proliferation activity of T cells derived from anormal healthy person “donor A” in the assay for the activity oftransducing costimulatory signal by various mouse anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with mouse anti-human AILIM monoclonalantibody.

[0349] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of mouse anti-humanAILIM monoclonal antibody.

[0350]FIG. 16 shows proliferation activity of T cells derived from anormal healthy person “donor A” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0351] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0352]FIG. 17 shows proliferation activity of T cells derived from anormal healthy person “donor B” in the assay for the activity oftransducing costimulatory signal by various mouse anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with mouse anti-human AILIM monoclonalantibody.

[0353] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of mouse anti-humanAILIM monoclonal antibody.

[0354] In this figure, “JHC1” indicated result of assay in whichanti-human CETP monoclonal antibody was used as the negative control,instead of the mouse anti-human AILIM monoclonal antibody.

[0355]FIG. 18 shows proliferation activity of T cells derived from anormal healthy person “donor B” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0356] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0357] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0358]FIG. 19 shows proliferation activity of T cells derived from anormal healthy person “donor B” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0359] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0360] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0361]FIG. 20 shows proliferation activity of T cells derived from anormal healthy person “donor C” in the assay for the activity oftransducing costimulatory signal by various mouse anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with mouse anti-human AILIM monoclonalantibody.

[0362] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of mouse anti-humanAILIM monoclonal antibody.

[0363] In this figure, “JHC1” indicates result of assay in whichanti-human CETP monoclonal antibody was used as the negative control,instead of the mouse anti-human AILIM monoclonal antibody.

[0364]FIG. 21 shows proliferation activity of T cells derived from anormal healthy person “donor C” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0365] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0366] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0367] Other notations are as follows:

[0368] “124”: human anti-human AILIM monoclonal antibody JMab124.

[0369] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0370] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0371]FIG. 22 shows proliferation activity of T cells derived from anormal healthy person “donor C” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0372] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0373] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0374] Other notations are as follows:

[0375] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0376] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0377] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0378]FIG. 23 shows proliferation activity of T cells derived from anormal healthy person “donor C” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0379] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0380] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0381] Other notations are as follows:

[0382] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0383] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0384] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0385]FIG. 24 shows proliferation activity of T cells derived from anormal healthy person “donor C” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0386] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0387] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0388] Other notations are as follows:

[0389] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0390] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0391]FIG. 25 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity oftransducing costimulatory signal by various mouse anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with mouse anti-human AILIM monoclonalantibody.

[0392] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of mouse anti-humanAILIM monoclonal antibody.

[0393] In this figure, “JHC1” indicates result of assay in whichanti-human CETP monoclonal antibody was used as the negative control,instead of the mouse anti-human AILIM monoclonal antibody.

[0394]FIG. 26 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0395] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0396] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0397] Other notations are as follows:

[0398] “124”: human anti-human AILIM monoclonal antibody JMab124.

[0399] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0400] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0401]FIG. 27 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0402] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of mouse anti-humanAILIM monoclonal antibody.

[0403] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0404] Other notations are as follows:

[0405] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0406] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0407] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0408]FIG. 28 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0409] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0410] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0411] Other notations are as follows:

[0412] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0413] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0414] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0415]FIG. 29 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0416] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0417] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0418] Other notations are as follows:

[0419] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0420] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0421]FIG. 30 shows proliferation activity of T cells derived from anormal healthy person “donor E” in the assay for the activity oftransducing costimulatory signal by various mouse anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with mouse anti-human AILIM monoclonalantibody.

[0422] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of mouse anti-humanAILIM monoclonal antibody.

[0423] In this figure, “JHC1” indicates result of assay in whichanti-human CETP monoclonal antibody was used as the negative control,instead of the mouse anti-human AILIM monoclonal antibody.

[0424]FIG. 31 shows proliferation activity of T cells derived from anormal healthy person “donor E” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0425] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0426] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0427] Other notations are as follows:

[0428] “124”: human anti-human AILIM monoclonal antibody JMab124.

[0429] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0430] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0431]FIG. 32 shows proliferation activity of T cells derived from anormal healthy person “donor E” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0432] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0433] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0434] Other notations are as follows:

[0435] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0436] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0437] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0438]FIG. 33 shows proliferation activity of T cells derived from anormal healthy person “donor E” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0439] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0440] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0441] Other notations are as follows:

[0442] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0443] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0444] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0445]FIG. 34 shows proliferation activity of T cells derived from anormal healthy person “donor E” in the assay for the activity oftransducing costimulatory signal by various human anti-human AILIMmonoclonal antibodies using a microplate coated with anti-human CD3monoclonal antibody together with human anti-human AILIM monoclonalantibody.

[0446] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0447] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0448] Other notations are as follows:

[0449] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0450] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0451]FIG. 35 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity of variousmouse anti-human AILIM monoclonal antibodies to transduce costimulatorysignal, when a solution of mouse anti-human human AILIM monoclonalantibody (in liquid phase) was added alone to a microplate coated withanti-human CD3 monoclonal antibody.

[0452] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of mouse anti-humanAILIM monoclonal antibody.

[0453] In this figure, “JHC1” indicates result of assay in whichanti-human CETP monoclonal antibody was used as the negative control,instead of the mouse anti-human AILIM monoclonal antibody.

[0454]FIG. 36 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity of varioushuman anti-human AILIM monoclonal antibodies to transduce costimulatorysignal when a solution of human anti-human AILIM monoclonal antibody (inliquid phase) was added alone to a microplate coated with anti-human CD3monoclonal antibody.

[0455] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0456] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0457] Other notations are as follows:

[0458] “124”: human anti-human AILIM monoclonal antibody JMab124.

[0459] “125”: human anti-human AILIM monoclonal antibody JMab125.

[0460] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0461]FIG. 37 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity of varioushuman anti-human AILIM monoclonal antibodies to transduce costimulatorysignal when a solution of human anti-human AILIM monoclonal antibody (inliquid phase) was added alone to a microplate coated with anti-human CD3monoclonal antibody.

[0462] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0463] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0464] Other notations are as follows:

[0465] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0466] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0467] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0468]FIG. 38 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity of varioushuman anti-human AILIM monoclonal antibodies to transduce costimulatorysignal when a solution of human anti-human AILIM monoclonal antibody (inliquid phase) was added alone to a microplate coated with anti-human CD3monoclonal antibody.

[0469] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0470] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0471] Other notations are as follows:

[0472] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0473] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0474] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0475]FIG. 39 shows proliferation activity of T cells derived from anormal healthy person “donor D” in the assay for the activity of varioushuman anti-human AILIM monoclonal antibodies to transduce costimulatorysignal when a solution of human anti-human AILIM monoclonal antibody (inliquid phase) was added alone to a microplate coated with anti-human CD3monoclonal antibody.

[0476] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of human anti-humanAILIM monoclonal antibody.

[0477] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0478] Other notations are as follows:

[0479] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0480] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0481]FIG. 40 shows the amount of IFN-γ produced in the culturesupernatant of T cells derived from a normal healthy person “donor B,”which were cultured in a microplate coated with mouse anti-human AILIMmonoclonal antibody together with anti-human CD3 monoclonal antibody.

[0482] The vertical axis indicates the concentration of IFN-g, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

[0483] In this figure, “JHC1” indicates result of assay in whichanti-human CETP monoclonal antibody was used as the negative control,instead of the mouse anti-human AILIM monoclonal antibody.

[0484]FIG. 41 shows the amount of IFN-g produced in the culturesupernatant of T cells derived from a normal healthy person “donor B,”which were cultured in a microplate coated with human anti-human AILIMmonoclonal antibody together with anti-human CD3 monoclonal antibody.

[0485] The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

[0486] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0487]FIG. 42 shows the amount of IFN-γ produced in the culturesupernatant of T cells derived from a normal healthy person “donor B,”which were cultured in a microplate coated with human anti-human AILIMmonoclonal antibody together with anti-human CD3 monoclonal antibody.

[0488] The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

[0489] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0490]FIG. 43 shows the amount of IFN-γ produced in the culturesupernatant of T cells derived from a normal healthy person “donor C,”which were cultured in a microplate coated with mouse anti-human AILIMmonoclonal antibody together with anti-human CD3 monoclonal antibody.

[0491] The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

[0492] In this figure, “JHC1” indicates result of assay in whichanti-human CETP monoclonal antibody was used as the negative control,instead of the mouse anti-human AILIM monoclonal antibody.

[0493]FIG. 44 shows the amount of IFN-γ produced in the culturesupernatant of T cells derived from a normal healthy person “donor C,”which were cultured in a microplate coated with human anti-human AILIMmonoclonal antibody together with anti-human CD3 monoclonal antibody.

[0494] The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

[0495] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0496] Other notations are as follows:

[0497] “124”: human anti-human AILIM monoclonal antibody JMab124.

[0498] “125”: human anti-human AILIM monoclonal antibody JMab125.

[0499] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0500]FIG. 45 shows the amount of IFN-γ produced in the culturesupernatant of T cells derived from a normal healthy person “donor C,”which were cultured in a microplate coated with human anti-human AILIMmonoclonal antibody together with anti-human CD3 monoclonal antibody.

[0501] The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

[0502] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0503] Other notations are as follows:

[0504] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0505] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0506] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0507]FIG. 46 shows the amount of IFN-γ produced in the culturesupernatant of T cells derived from a normal healthy person “donor C,”which were cultured in a microplate coated with human anti-human AILIMmonoclonal antibody together with anti-human CD3 monoclonal antibody.

[0508] The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

[0509] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0510] Other notations are as follows:

[0511] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0512] “138”: human anti-human AILIM monoclonal antibody JMab238.

[0513] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0514]FIG. 47 shows the amount of IFN-γ produced in the culturesupernatant of T cells derived from a normal healthy person “donor C,”which were cultured in a microplate coated with human anti-human AILIMmonoclonal antibody together with anti-human CD3 monoclonal antibody.

[0515] The vertical axis indicates the concentration of IFN-γ, and thehorizontal axis indicates the concentration of the mouse anti-humanAILIM monoclonal antibody.

[0516] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0517] Other notations are as follows:

[0518] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0519] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0520]FIG. 48 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor A”, withPBMC of a normal healthy person “donor D” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

[0521] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0522] Each description in the figures shows the following.

[0523] “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86antibody “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0524] “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

[0525] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0526]FIG. 49 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor A”, withPBMC of a normal healthy person “donor D” by various human anti-humanAILIM monoclonal antibodies in the proliferation test of the T cellsthrough the mixed lymphocyte reactions (MLR).

[0527] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0528] Other notations are as follows:

[0529] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0530] “JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

[0531] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0532] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0533] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0534] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0535] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0536] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0537]FIG. 50 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor D”, withPBMC of a normal healthy person “donor B” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

[0538] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0539] Each description in the figures shows the following.

[0540] “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0541] “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0542] “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

[0543] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0544]FIG. 51 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor D”, withPBMC of a normal healthy person “donor B” by various human anti-humanAILIM monoclonal antibodies in the proliferation test of the T cellsthrough the mixed lymphocyte reactions (MLR).

[0545] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0546] Other notations are as follows:

[0547] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0548] “JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

[0549] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0550] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0551] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0552] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0553] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0554] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0555]FIG. 52 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor C”, withPBMC of a normal healthy person “donor A” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

[0556] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0557] Each description in the figures shows the following.

[0558] “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0559] “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0560] “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

[0561] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0562]FIG. 53 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor C”, withPBMC of a normal healthy person “donor A” by various human anti-humanAILIM monoclonal antibodies in the proliferation test of the T cellsthrough the mixed lymphocyte reactions (MLR).

[0563] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0564] Other notations are as follows:

[0565] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0566] “JMab-124”: human anti-human AILIM monoclonal antibody JMab 124.

[0567] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0568] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0569] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0570] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0571] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0572] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0573]FIG. 54 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor E”, withPBMC of a normal healthy person “donor G” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

[0574] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0575] Each description in the figures shows the following.

[0576] “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0577] “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0578] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0579] “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

[0580]FIG. 55 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor E”, withPBMC of a normal healthy person “donor G” by various human anti-humanAILIM monoclonal antibodies in the proliferation test of the T cellsthrough the mixed lymphocyte reactions (MLR).

[0581] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0582] Other notations are as follows:

[0583] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0584] “JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

[0585] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0586] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0587] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0588] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0589]FIG. 56 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor F”, withPBMC of a normal healthy person “donor E” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

[0590] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0591] Each description in the figures shows the following.

[0592] “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0593] “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0594] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0595] “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

[0596]FIG. 57 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor F”, withPBMC of a normal healthy person “donor E” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

[0597] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0598] Each description in the figures shows the following.

[0599] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0600] “JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

[0601] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0602] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0603] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0604] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0605]FIG. 58 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor G”, withPBMC of a normal healthy person “donor F” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

[0606] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0607] Each description in the figures shows the following.

[0608] “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0609] “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0610] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0611] “CTLA4-Ig”: Human CTLA4-IgFc chimeric molecule

[0612]FIG. 59 shows the inhibitory effect on T cell proliferation in thecase of culturing T cells from a normal healthy person “donor G”, withPBMC of a normal healthy person “donor F” by various test samples in theproliferation test of the T cells through the mixed lymphocyte reactions(MLR).

[0613] The vertical axis indicates the amount of incorporation of[³H]thymidine as an index showing a level of cell proliferation, and thehorizontal axis shows the concentration of the test samples.

[0614] Each description in the figures shows the following.

[0615] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0616] “JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

[0617] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0618] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0619] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0620] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0621]FIG. 60 shows the inhibitory effect of various control testsubstances on the proliferation of T cells in the assay using mixedlymphocyte reaction (MLR). T cells from a normal healthy person “donorA” were co-cultured with PBMCs from a normal healthy person “donor D”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

[0622] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0623] Each description in the figures shows the following.

[0624] “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0625] “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0626] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0627]FIG. 61 shows the inhibitory effect of various human anti-humanAILIM monoclonal antibodies on the proliferation of T cells in the assayusing mixed lymphocyte reaction (MLR). T cells from a normal healthyperson “donor A” were co-cultured with PBMCs from a normal healthyperson “donor D” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

[0628] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0629] Other notations are as follows:

[0630] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0631] “JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

[0632] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0633] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0634] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0635] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0636] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0637] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0638]FIG. 62 shows the inhibitory effect of various control testsubstances on the proliferation of T cells in the assay using mixedlymphocyte reaction (MLR). T cells from a normal healthy person “donorD” were co-cultured with PBMCs from a normal healthy person “donor B”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

[0639] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0640] Each description in the figures shows the following.

[0641] “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0642] “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0643] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0644]FIG. 63 shows the inhibitory effect of various human anti-humanAILIM monoclonal antibodies on the proliferation of T cells in the assayusing mixed lymphocyte reaction (MLR). T cells from a normal healthyperson “donor D” were co-cultured with PBMCs from a normal healthyperson “donor B” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

[0645] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0646] Other notations are as follows:

[0647] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0648] “JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

[0649] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0650] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0651] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0652] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0653] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0654] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0655]FIG. 64 shows the inhibitory effect of various control testsubstances on the proliferation of T cells in the assay using mixedlymphocyte reaction (MLR). T cells from a normal healthy person “donorC” were co-cultured with PBMCs from a normal healthy person “donor A”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

[0656] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0657] Each description in the figures shows the following.

[0658] “CD80+86”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0659] “mIgG1”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0660] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0661]FIG. 65 shows the inhibitory effect of various human anti-humanAILIM monoclonal antibodies on the proliferation of T cells in the assayusing mixed lymphocyte reaction (MLR). T cells from a normal healthyperson “donor C” were co-cultured with PBMCs from a normal healthyperson “donor A” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

[0662] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0663] Other notations are as follows:

[0664] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0665] “JMab-124”: human anti-human AILIM monoclonal antibody JMab124.

[0666] “126”: human anti-human AILIM monoclonal antibody JMab126.

[0667] “127”: human anti-human AILIM monoclonal antibody JMab127.

[0668] “128”: human anti-human AILIM monoclonal antibody JMab128.

[0669] “135”: human anti-human AILIM monoclonal antibody JMab135.

[0670] “136”: human anti-human AILIM monoclonal antibody JMab136.

[0671] “137”: human anti-human AILIM monoclonal antibody JMab137.

[0672]FIG. 66 shows the inhibitory effect of various control testsubstances on the proliferation of T cells in the assay using mixedlymphocyte reaction (MLR). T cells from a normal healthy person “donorE” were co-cultured with PBMCs from a normal healthy person “donor G”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

[0673] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0674] Each description in the figures shows the following.

[0675] “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0676] “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0677] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0678]FIG. 67 shows the inhibitory effect of various human anti-humanAILIM monoclonal antibodies on the proliferation of T cells in the assayusing mixed lymphocyte reaction (MLR). T cells from a normal healthyperson “donor E” were co-cultured with PBMCs from a normal healthyperson “donor G” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

[0679] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0680] Other notations are as follows:

[0681] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0682] “JMab-136”: human anti-human AILIM monoclonal antibody JMab 136.

[0683] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0684] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0685] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0686] “141”: human anti-human AILIM monoclonal antibody JMab141.

[0687]FIG. 68 shows the inhibitory effect of various control testsubstances on the proliferation of T cells in the assay using mixedlymphocyte reaction (MLR). T cells from a normal healthy person “donorG” were co-cultured with PBMCs from a normal healthy person “donor F”pre-cultured in the presence of human CTLA4-Ig chimeric molecule.

[0688] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0689] Each description in the figures shows the following.

[0690] “control mIgG”: Anti-human CD34/IgG1 mouse monoclonal antibody

[0691] “CD80+86 Ab”: The mixture of anti-CD80 antibody and anti-CD86antibody

[0692] “SA12”: Anti-human AILIM mouse monoclonal antibody

[0693]FIG. 69 shows the inhibitory effect of various human anti-humanAILIM monoclonal antibodies on the proliferation of T cells in the assayusing mixed lymphocyte reaction (MLR). T cells from a normal healthyperson “donor G” were co-cultured with PBMCs from a normal healthyperson “donor F” pre-cultured in the presence of human CTLA4-Ig chimericmolecule.

[0694] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentrations of test substances.

[0695] Other notations are as follows:

[0696] “anti-KLH”: human anti-KLH monoclonal antibody as a negativecontrol.

[0697] “JMab-136”: human anti-human AILIM monoclonal antibody JMab136.

[0698] “138”: human anti-human AILIM monoclonal antibody JMab138.

[0699] “139”: human anti-human AILIM monoclonal antibody JMab139.

[0700] “140”: human anti-human AILIM monoclonal antibody JMab140.

[0701] “141”: human anti-human AILIM monoclonal antibody JMab 141.

[0702]FIG. 70 shows ADCC-inducing activity of various human anti-humanAILIM monoclonal antibodies and control antibodies where wild-type CHOcells were used as the target cells.

[0703] The vertical axis indicates the rate of cytotoxicity caused byADCC-inducing activity of antibody, and the horizontal axis indicatesthe concentration of antibody.

[0704]FIG. 71 shows ADCC-inducing activity of various human anti-humanAILIM monoclonal antibodies and control antibody where humanAILIM-overexpressing recombinant CHO cells were used as the targetcells.

[0705] The vertical axis indicates the frequency of cell damage resultedfrom the ADCC-inducing activity of antibody, and the horizontal axisindicates the concentration of antibody.

[0706]FIG. 72 shows the inhibitory effect of anti-AILIM antibody ondelayed allergy.

[0707] The vertical axis indicates the size of redness measured as anindex of the onset of delayed allergy, and the horizontal axis indicatesthe type of test sample given to animal subjects.

[0708]FIG. 73 shows proliferation activity of monkey T cells in theassay to determine the activity of various human anti-human AILIMmonoclonal antibodies to transduce costimulatory signal, using amicroplate coated with human anti-human AILIM monoclonal antibodytogether with anti-human CD3 monoclonal antibody.

[0709] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of the human anti-humanAILIM monoclonal antibody.

[0710] In this figure, “anti-KLH” indicates result of assay in whichhuman anti-KLH monoclonal antibody was used as the negative control,instead of the human anti-human AILIM monoclonal antibody.

[0711]FIG. 74 shows inhibitory activity of the negative control antibodyagainst binding between soluble AILIM ligands (hB7h-IgFc) and solubleAILIM (AILIM-IgFc) of various concentrations.

[0712] The vertical axis indicates absorbance as an index for theinhibitory activity, and the horizontal axis indicates the concentrationof soluble AILIM.

[0713]FIG. 75 shows inhibitory activity of anti AILIM antibody againstbinding between soluble AILIM ligands (hB7h-IgFc) and soluble AILIM(AILIM-IgFc) of various concentrations.

[0714] The vertical axis indicates absorbance as an index for theinhibitory activity, and the horizontal axis indicates the concentrationof soluble AILIM.

[0715]FIG. 76 shows inhibitory activity of anti AILIM antibody atvarious concentrations against binding between soluble AILIM ligands(hB7h-IgFc) and soluble AILIM (AILIM-IgFc).

[0716] The vertical axis indicates absorbance as an index for theinhibitory activity, and the horizontal axis indicates the concentrationof soluble AILIM.

[0717]FIG. 77 shows inhibitory activity of various human anti-humanAILIM monoclonal antibodies to human T cell proliferation in the assayto determine the activity of transducing costimulatory signal using amicroplate coated with soluble human AILIM ligand (hB7h-IgFc) togetherwith anti-human CD3 monoclonal antibody.

[0718] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of antibody.

[0719]FIG. 78 shows inhibitory activity of various human anti-humanAILIM monoclonal antibodies to monkey T cell proliferation in the assayto determine the activity of transducing costimulatory signal using amicroplate coated with soluble human AILIM ligand (hB7h-IgFc) togetherwith anti-human CD3 monoclonal antibody.

[0720] The vertical axis indicates the amount of cellular incorporationof [³H]thymidine as an index of the degree of cell proliferation, andthe horizontal axis indicates the concentration of antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0721] The present inventions are described in detail herein below bydefining terminologies of the present invention.

[0722] Herein, “mammal” means human, bovine, goat, rabbit, mouse, rat,hamster, and guinea pig; preferred is human, rabbit, rat, hamster, ormouse and particularly preferred is human, rat, hamster, or mouse.

[0723] The term “mammals other than humans” and “non-human mammals” usedherein, are synomic to each other, meaning all mammals other than humansdefined above.

[0724] The term “amino acids” used herein, means every amino acidexisting in nature, preferably those described according to thealphabetical three letters system or single letter system as shownbelow:

[0725] glycine (Gly/G), alanine (Ala/A), valine (Val/V), leucine(Leu/L), isoleucine (Ile/I), serine (Ser/S), threonine (Thr/T), asparticacid (Asp/D), glutamic acid (Glu/E), asparagine (Asn/N), glutamine(Gln/Q), lysine (Lys/K), arginine (Arg/R), cysteine (Cys/C), methionine(Met/M), phenylalanine (Phe/F), tyrosine (Tyr/Y), tryptophan (Trp/W),histidine (His/H), proline (Pro/P).

[0726] The term “AILIM” used herein is the abbreviation for ActivationInducible Lymphocyte Immunomodulatory Molecule, indicating a mammaliancell surface molecule having the structure and function as alreadydescribed in a previous report, more preferably a human-derived AILIM inparticular (for example, International Immunology, Vol. 12, No. 1,p.51-55; GenBank Accession Number: BAA82129 (human), BAA82128 (rat),BAA82127 (rat variant), and BAA82126 (mouse)).

[0727] Alternatively, this AILIM is also referred to as ICOS (UnexaminedPublished Japanese Patent Application (JP-A) No. Hei 11-29599,International Patent Application No. WO98/38216), and theseabbreviations indicate the same molecule.

[0728] “AILIM ligand” used herein means a cell surface molecule whichinteracts with said co-stimulatory molecule AILIM (ICOS), and isreferred to as B7h, B7RP-1, GL50 or LICOS (Nature, Vol. 402, No. 6763,p.827-832, 1999; Nature Medicine, Vol. 5, No. 12, p.1365-1369, 1999; J.Immunology, Vol. 164, p.1653-1657, 2000; Curr. Biol. Vol. 10, No. 6,p.333-336, 2000).

[0729] Moreover, “AILIM” used herein also includes a polypeptide havingsubstantially the same amino acid sequence as that of AILIM of eachmammal described in the references, and particularly preferably, that ofhuman AILIM. Furthermore, a human AILIM variant which is similar to therat AILIM variant already reported (GenBank Accession Number: BAA82127)is also included in “AILIM” of this invention.

[0730] “AILIM ligand” used herein is also defined to have a similarmeaning as above.

[0731] Herein, “polypeptides having essentially identical amino acidsequence” means variant polypeptides as described below.

[0732] That is, as long as these variant polypeptides have biologicalproperties essentially equivalent to the natural type AILIM(particularly preferably the human-derived AILIM), they are polypeptidesof this invention. Like those having amino acid sequence of the naturaltype AILIM, in which a plurality of amino acid residues, preferably 1 to10 amino acid residues, most preferably 1 to 5 amino acid residues aredeleted and/or modified, and to which a plurality of amino acidresidues, preferably 1 to 10 amino acid residues, most preferably 1 to 5amino acid residues are added.

[0733] Furthermore, they may be variant polypeptides having plurality ofthese substitution, deletion, modification and addition of amino acidresidues in the molecule.

[0734] “AILIM ligand” in this invention is also defined to have asimilar meaning as above.

[0735] AILIM (particularly human AILIM) and AILIM ligand (particularlyhuman AILIM ligand) in this invention can be prepared by, in addition togene recombinant technique, appropriately using well-known methods inthis technical field such as chemical synthesis method, cell culturemethod, etc. or these methods with modifications.

[0736] Such substitution, deletion, or insertion of amino acids can beachieved according to the usual method (Experimental Medicine:SUPPLEMENT, “Handbook of Genetic Engineering” (1992); and so on).

[0737] Examples of methods for producing mutant polypeptides asmentioned above are synthetic oligonucleotide site-directed mutagenesis(gapped duplex method), point mutagenesis by which a point mutation isintroduced at random by treatment with nitrite or sulfite, the method bywhich a deletion mutant is prepared with Bal31 enzyme and the like,cassette mutagenesis, linker scanning method, miss incorporation method,mismatch primer method, DNA segment synthesis method, etc.

[0738] Synthetic oligonucleotide site-directed mutagenesis (gappedduplex method) can be, for example, performed as follows. The regiondesired to be mutagenized is cloned into M13 phage vector having ambermutation to prepare the single-stranded phage DNA. After RF I DNA of M13vector without amber mutation is linearized by restriction enzymetreatment, DNA is mixed with the single-stranded phage DNA mentionedabove, denatured, and annealed thereby forming “gapped duplex DNA.” Asynthetic oligonucleotide into which mutations are introduced ishybridized with the gapped duplex DNA and the closed-circulardouble-stranded DNAs are prepared by the reactions with DNA polymeraseand DNA ligase. E. coli mutS cells, deficient in mismatch repairactivity, are transfected with this DNA. E. coli cells withoutsuppressor activity are infected with the grown phages, and only phageswithout amber mutation are screened.

[0739] The method by which a point mutation is introduced with nitriteutilizes, for example the principle as mentioned below. If DNA istreated with nitrite, bases are deaminated to change adenine intohypoxanthine, cytosine into uracil, and guanine into xanthine. Ifdeaminated DNA is introduced into cells, “A:T” and “G:C” are replacedwith “G:C” and “A:T”, respectively, because hypoxanthine, uracil, andxanthine form a base pair with cytosine, adenine, and thymine,respectively, in the DNA replication. Actually, single-stranded DNAfragments treated with nitrite are hybridized with “gapped duplex DNA”,and thereafter mutant strains are separated by manipulating in the sameway as synthetic oligonucleotide site-directed mutagenesis (gappedduplex method).

[0740] In addition, “AILIM” herein also includes “a portion” of saidAILIM. Herein, “a portion” means a polypeptide comprising any partialsequence of the above-defined AILIM amino acid sequence.

[0741] Preferably, said portion indicates the extracellular region ofabove-defined AILIM (particularly preferably a human AILIM) or anyportion thereof.

[0742] “AILIM ligand” in this invention is also defined to have asimilar meaning as above.

[0743] “Portion” of said AILIM (preferably the extracellular region ofAILIM or any portion thereof) can be prepared according to well-knownmethods in this technical field as described below or according to theirmodified methods by genetic recombination technique or chemicalsynthesis method, or by suitably cleaving AILIM (particularly preferablya human AILIM) isolated by cell culture method using proteolyticenzymes, etc.

[0744] “Portion of AILIM ligand” can be also prepared by similar methodsas described above.

[0745] “Human antibody” of this invention is a human antibody whichbinds to the above-defined AILIM or a portion thereof (particularlypreferably a human-derived AILIM or a portion thereof). Specifically, itmeans a human-derived polyclonal antibody (human polyclonal antibody,human antiserum) or human-derived monoclonal antibody (human monoclonalantibody).

[0746] “Human monoclonal antibody” of this invention is a humanmonoclonal antibody which binds to the above-defined AILIM or a portionthereof (particularly preferably a human-derived AILIM or a portionthereof).

[0747] More specifically, all the regions comprising the variable andconstant regions of the heavy chain (H-chain), and the variable andconstant regions of the light chain (L-chain) consist of humanimmunoglobulin derived from gene encoding said human immunoglobulin.L-chain is exemplified by human κ chain or human λ chain.

[0748] Human monoclonal antibody which binds to AILIM (particularlypreferably a human-derived AILIM) of this invention or a portion thereofis a human monoclonal antibody having characteristic defined in any ofaforementioned (5) through (42) or (84).

[0749] More specifically, it includes various human monoclonalantibodies having various characteristics and industrial applicabilityas described in examples and drawings below.

[0750] A preferred embodiment of human monoclonal antibody of thisinvention is a human monoclonal antibody which binds to AILIM or aportion thereof defined in any of aforementioned (5) through (42) or(84).

[0751] Most preferable embodiment is a human monoclonal antibody whichbinds to human AILIM as described in (30) or (39).

[0752] “Human monoclonal antibody” of this invention can be prepared byimmunizing following transgenic non-human mammals producing humanantibody with any of the immunogens (antigens) described below.

[0753] (a) a natural cell or artificially established cell lineexpressing aforementioned AILIM (particularly preferably a human-derivedAILIM) on the cell surface;

[0754] (b) a genetic recombinant cell prepared using geneticrecombination techniques so as to express above-defined AILIM(particularly preferably a human-derived AILIM) on the cell surface;

[0755] (c) a cell lysate obtained by solubilizing cells aforementionedin (a) or (b), or a polypeptide fragment of AILIM (particularlypreferably a human-derived AILIM) purified from said cell lysate;

[0756] (d) a genetic recombinant cell prepared using geneticrecombination techniques so as to express a portion (particularlypreferably the extracellular region or any preferable peptide thereof)of above-defined AILIM (particularly preferably a human-derived AILIM)as a soluble polypeptide;

[0757] (e) a culture supernatant obtained by culturing the geneticrecombinant cell aforementioned in (d) or an extracellular regionpolypeptide (soluble AILIM) of AILIM (particularly preferably ahuman-derived AILIM) purified from said culture supernatant; or

[0758] (f) a portion (particularly preferably the extracellular regionor any preferable peptide thereof) of chemically synthesized AILIM(particularly preferably a human-derived AILIM).

[0759] Furthermore, monoclonal antibody of this invention can be alsoobtained from culture supernatant by culturing a “genetic recombinanthost” [herein, said host is an eukaryotic cell other than fertilizedeggs (preferably mammalian cells such as CHO, lymphocytes, and myelomacells)], which can be prepared by transforming a host with cDNAs(preferably a vector containing said cDNAs) encoding each of the heavyand light chains of such a human monoclonal antibody of this inventionusing genetic recombination techniques, and which produces geneticrecombinant human monoclonal antibody.

[0760] Specifically, the monoclonal antibody of this invention can beobtained by culturing genetic recombinant host described in any ofaforementioned (60) through (62) or (64) through (80) of this invention(herein, said host is an eukaryotic cell other than a fertilized egg(preferably mammalian cells such as CHO, lymphocytes, and myelomacells)).

[0761] In addition, human monoclonal antibody of this invention may be ahuman monoclonal antibody having any isotype belonging to IgG (IgG1,IgG2, IgG3 and IgG4), IgH, IgA (IgA1 and IgA2), IgD or IgE. Preferably,said monoclonal antibody belongs to IgG (IgG1, IgG2, IgG3 and IgG4),more preferably IgG1, IgG2 or IgG4.

[0762] Human monoclonal antibody of this invention can be prepared byimmunizing transgenic non-human mammal producing human antibody such ashuman antibody-producing transgenic mouse described below with any ofthe immunogens (antigens) aforementioned in (a) through (f) according toknown commonly used manufacturing method.

[0763] That is, for example, said transgenic non-human mammal producinghuman antibody is immunized with said antigen in combination withFreund's adjuvant as the occasion demands. Polyclonal antibody can beobtained from sera collected from said immunized animal. Monoclonalantibody can be manufactured by preparing fusion cells (hybridomas) fromsaid antibody-producing cells isolated from said immunized animal andmyeloma cells with no autoantibody-producing ability, and cloning saidhybridomas to select a clone producing the monoclonal antibody with aspecific affinity to the antigen used for immunizing the mammal.

[0764] More specifically, monoclonal antibody can be prepared asdescribed below. That is, said human antibody-producing transgenicnon-human mammal (particularly preferably “human antibody-producingtransgenic mouse”) is immunized by injecting any of the immunogensaforementioned in (a) through (c) intradermally, intramuscularly,intravenously, into the footpad, or intraperitoneally once to severaltimes, or transplanting said immunogen into said mammal. Usually,immunizations are performed once to four times every one to fourteendays after the first immunization. Antibody-producing cells are obtainedfrom the mammal so immunized in about one to five days after the lastimmunization. The frequency and interval of immunizations can beappropriately arranged depending on, e.g., property of the immunogenused.

[0765] Hybridomas that secrete a human monoclonal antibody can beprepared by the method of Köhler and Milstein (Nature, Vol. 256,pp.495-497 (1975)) and by its modified method. Namely, hybridomas areprepared by fusing antibody-producing cells contained in a spleen, lymphnode, bone marrow, or tonsil obtained from the human antibody-producingtransgenic non-human mammal immunized as mentioned above, preferably aspleen, with myelomas without autoantibody-producing ability, which arederived from, preferably, a mammal such as a mouse, rat, guinea pig,hamster, rabbit, or human, or more preferably, a mouse, rat, or human.

[0766] For example, mouse-derived myeloma P3/X63-AG8.653 (ATCC No.CRL-1580), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14(Sp2/0, Sp2), NSO, PAI, F0, or BW5147, rat-derived myeloma210RCY3-Ag.2.3., or human-derived myeloma U-266AR1, GM1500-6TG-A1-2,UC729-6, CEM-AGR, D1R11, or CEM-T15 can be used as a myeloma used forthe cell fusion.

[0767] Cells producing monoclonal antibodies (for example, hybridomas)can be screened by cultivating the cells, for example, in microtiterplates and by measuring the reactivity of the culture supernatant in thewell in which hybridoma growth is observed, to the immunogen used forthe immunization mentioned above, for example, by enzyme immunoassaysuch as radio immunoassay (RIA) and enzyme-linked immuno-solvent assay(ELISA).

[0768] The monoclonal antibodies can be produced from hybridomas bycultivating the hybridomas in vitro or in vivo such as in the ascitesfluid of a mouse, rat, guinea pig, hamster, or rabbit, preferably amouse or rat, more preferably mouse and isolating the antibodies fromthe resulting the culture supernatant or ascites fluid of a mammal.

[0769] Monoclonal antibodies of this invention can be manufactured on alarge scale by the following method:

[0770] (1) genes (cDNAs, etc.) encoding each of the heavy and lightchains of said monoclonal antibody are cloned from said hybridomas;

[0771] (2) cloned genes encoding each of the heavy and light chains areinserted into separate vectors or a single vector to prepare theexpression vector;

[0772] (3) said expression vector is transferred into a fertilized eggof a desired non-human mammal (such as goat);

[0773] (4) said fertilized egg transferred with the gene is transplantedinto the uterus of a foster mother to obtain a chimeric non-humananimal;

[0774] (5) by further mating said chimeric goat with another non-humanmammal, a transgenic non-human mammal (cattle, goat, sheep or swine)with genes encoding each of said heavy and light chains incorporatedinto the endogenous gene is produced; and

[0775] (6) from the milk of said transgenic non-human mammal, monoclonalantibody derived from said human monoclonal antibody gene is obtained ona large scale Nikkei Science, April, 1997, p.78-84).

[0776] Cultivating in vitro the cells producing the monoclonalantibodies can be performed depending on, e.g., the property of cells tobe cultured, the object of a test study, and the various conditions of acultivating method, by using known nutrient media or any nutrient mediaderived from known basal media for growing, maintaining, and storing thehybridomas to produce monoclonal antibodies in culture supernatant.

[0777] Examples of basal media are low calcium concentration media suchas Ham′F12 medium, MCDB153 medium, or low calcium concentration MEMmedium, and high calcium concentration media such as MCDB104 medium, MEMmedium, D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. Thebasal media can contain, for example, sera, hormones, cytokines, and/orvarious inorganic or organic substances depending on the objective.

[0778] Monoclonal antibodies can be isolated and purified from theculture supernatant or ascites fluid mentioned above by saturatedammonium sulfate precipitation, euglobulin precipitation method, caproicacid method, caprylic acid method, ion exchange chromatography (DEAE orDE52), affinity chromatography using anti-immunoglobulin column orprotein A column.

[0779] Human monoclonal antibody of this invention includes humanmonoclonal antibodies consisting of the heavy chain and/or light chainof which amino acid sequence for each chain have one or more amino acidresidues deleted, substituted or added.

[0780] Herein, “more amino acid residues” means a plurality of aminoacids, specifically 1 to 10 amino acid residues, preferably 1 to 5 aminoacid residues.

[0781] A partial modification (deletion, substitution, insertion oraddition) as described above can be introduced into the amino acidsequence of human monoclonal antibody of this invention by partialalteration of base sequence encoding said amino acid sequence. Thispartial alteration of base sequence can be introduced by standard methodusing known site-specific mutagenesis technique (Proc. Natl. Acad. Sci.USA, Vol. 81, p.5662-5666, 1984).

[0782] “Transgenic human antibody-producing non-human mammal”,particularly human antibody-producing transgenic mouse which is apreferred embodiment, can be prepared according to published literature(Nature Genetics, Vol. 7, p.13-21, 1994; Nature Genetics, Vol. 15,p.146-156, 1997; Published Japanese Translation of InternationalPublication No. Hei 4-504365; Published Japanese Translation ofPublication No. Hei 7-509137; Nikkei Science, June, p.40-50, 1995;International Patent Publication No. WO94/25585; Nature, Vol. 368,p.856-859, 1994; and Published Japanese Translation of Publication No.Hei 6-500233, etc.)

[0783] Specifically, said human antibody-producing transgenic mice canbe prepared, for example, using techniques consisting of the followingprocesses:

[0784] (1) preparing a knockout mouse which endogenous immunoglobulinheavy chain gene is functionally inactivated by substituting at least aportion of gene locus of the mouse endogenous immunoglobulin heavy chainwith a drug tolerance marker gene (such as neomycin tolerance gene) byhomologous recombination;

[0785] (2) preparing a knockout mouse which endogenous immunoglobulinlight chain gene (particularly the κ chain gene) is functionallyinactivated by substituting at least a portion of gene locus of themouse endogenous immunoglobulin light chain with a drug tolerance markergene (such as neomycin tolerance gene) by homologous recombination;

[0786] (3) preparing a transgenic mouse which desired region of thehuman immunoglobulin heavy chain gene locus is incorporated into themouse chromosome using a vector represented by the yeast artificialchromosome (YAC) capable of carrying a giant gene;

[0787] (4) preparing a transgenic mouse which desired region of thehuman immunoglobulin light chain gene locus (particularly the κ chaingene) is incorporated into the mouse chromosome using a vectorrepresented by the yeast artificial chromosome (YAC) capable of carryinga giant gene; and

[0788] (5) preparing a transgenic mouse which endogenous immunoglobulinheavy and light chains gene loci are both functionally inactivated andwhich chromosome is incorporated with the desired regions of both of thehuman immunoglobulin heavy and light chains gene loci by mating knockoutand transgenic mice aforementioned in (1) through (4) in arbitraryorders.

[0789] The above-described knockout mouse can be prepared bysubstituting the suitable region of the mouse endogenous immunoglobulingene locus with a foreign marker gene (such as neomycin tolerance gene)based on homologous recombination to inactivate said gene locus so asnot to be rearranged. For the inactivation using said homologousrecombination, for example, a method referred to as positive negativeselection (PNS) can be used (Nikkei Science, May, p.52-62, 1994).

[0790] Functional inactivation of the immunoglobulin heavy chain genelocus can be achieved, for example, by introducing a lesion into a partof the J- or C-region (for example, Cμ region). And functionalinactivation of the immunoglobulin light chain (for example, κ chain)can be achieved, for example, by introducing a lesion into a part of J-or C-region, or a region extending over J- and C-regions.

[0791] A transgenic mouse can be prepared according to the method asusually used for producing a transgenic animal (for example, see “NewestManual of Animal Cell Experiment”, LIC press, Chapter 7, pp.361-408,(1990)). Specifically, for example, the HPRT-negative(hypoxanthine-guanine phosphoribosyltransferase gene deficient) ES cell(embryonic stem cell) derived from a normal mouse blastocyst is fusedwith yeast containing the YAC vector inserted with the gene encodingsaid human immunoglobulin heavy chain gene locus or light chain genelocus or a portion thereof and the HPRT gene using spheroplast fusionmethod. ES cells whose mouse endogenous gene is integrated with saidforeign gene are selected by HAT selection method. Then, the ES cellsscreened are microinjected into a fertilized egg obtained from anothernormal mouse (blastocyst) (Proc. Natl. Acad. Sci. USA, Vol.77, No.12,pp.7380-7384 (1980); U.S. Pat. No. 4,873,191). The blastocyst istransplanted into the uterus of another normal mouse as the fostermother. Then, chimeric transgenic mice are born from the foster mothermouse. By mating the chimeric transgenic mice with normal mice,heterogeneic transgenic mice are obtained. By mating the heterogeneictransgenic mice with each other, homogeneic transgenic mice are obtainedaccording to Mendel's laws.

[0792] The “portion of a monoclonal antibody” used in the presentinvention means a partial region of the above-mentioned human monoclonalantibody of the present invention, and specifically, includes F(ab′)₂,Fab′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulfidestabilized Fv), or dAb (single domain antibody) (Exp. Opin. Ther.Patents, Vol.6, No.5, pp.441-456 (1996)).

[0793] “F(ab′)₂” and “Fab′” can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andmeans an antibody fragment generated by digesting immunoglobulin nearthe disulfide bonds in the hinge regions existing between each of thetwo H chains. For example, papain cleaves IgG upstream of the disulfidebonds in the hinge regions existing between each of the two H chains togenerate two homologous antibody fragments in which an L chain composedof V_(L) (L chain variable region) and C_(L) (L chain constant region),and an H chain fragment composed of V_(H) (H chain variable region) andC_(H)γ1 (γ1 region in the constant region of H chain) are connected attheir C terminal regions through a disulfide bond. Each of such twohomologous antibody fragments is called Fab′. Pepsin also cleaves IgGdownstream of the disulfide bonds in the hinge regions existing betweeneach of the two H chains to generate an antibody fragment slightlylarger than the fragment in which the two above-mentioned Fab′ areconnected at the hinge region. This antibody fragment is called F(ab′)₂.

[0794] “Binding rate constant (ka)” herein means a value indicating thebinding strength (degree) of said monoclonal antibody to the targetantigen calculated based on the antibody antigen reaction kinetics.“Dissociation rate constant (kd)” means a value indicating thedissociation strength (degree) of said monoclonal antibody from thetarget antigen. “Dissociation constant (Kd)” is a value obtained bydividing said “dissociation rate constant (kd)” by said “binding rateconstant (ka)” value. These constants are used to represent the affinityof said monoclonal antibody to antigen and its activity to neutralizeantigen.

[0795] Said constants can be analyzed according to various methods, andcan be easily analyzed using a commercial assay kit BiacoreX (AmershamPharmacia) or a similar kit according to the manual and experimentalmethod attached to said kit. ka, kd and Kd values obtained using saidkit are expressed in 1/M.Sec, 1/Sec and M (mol) units, respectively.Higher ka values indicate stronger antigen binding activity ofmonoclonal antibody tested, and smaller Kd values show stronger antigenneutralizing activity of antibody.

[0796] Human monoclonal antibody of this invention includes those havingthe ka, kd or Kd value as shown in following (1) through (3):

[0797] (1) human monoclonal antibody which binds to human AILIM or aportion thereof with the binding rate constant (ka) of 1.0×10⁴ (1/M.Sec)or more, preferably 1.0×10⁵ (1/M.Sec) or more.

[0798] (2) human monoclonal antibody which binds to human AILIM or aportion thereof with the dissociation rate constant (kd) of 1.0×10⁻⁴(1/Sec) or less, preferably 1.0×10⁻⁵ (1/Sec) or less.

[0799] (3) human monoclonal antibody which has a reactivity to humanAILIM or a portion thereof with the dissociation constant (Kd) of1.0×10⁻⁷ (M) or less, preferably 1.0×10⁻⁸ (M) or less and morepreferably 1.0×10⁻⁹ (M) or less.

[0800] In this case, each value of ka, kd and Kd described above isexpected to slightly fluctuate depending on various conditions at thetime of measurement with a margin of error but with practically nofluctuation in indexes in general.

[0801] “Monoclonal antibody-producing cell” or genetic recombinant humanmonoclonal antibody-producing “genetic recombinant host” of thisinvention (herein, said host is a cell excluding fertilized egg) meansany cell producing aforementioned human monoclonal antibody of thisinvention.

[0802] Specifically, for example, it includes cells described in any offollowing (1) through (3), but is not limited to them:

[0803] (1) human monoclonal antibody-producing B cell obtained byimmunizing aforementioned human antibody-producing transgenic non-humanmammal with the above-defined immunogen (antigen) and collecting thecell from said immunized animal.

[0804] (2) aforementioned fusion cell (hybridoma) resulted by fusion ofthe human monoclonal antibody-producing B cell thus obtained with amyeloma cell derived from mammal.

[0805] (3) genetic recombinant human monoclonal antibody-producinggenetic recombinant cell obtained by transforming a cell excluding saidB cell and hybridoma (for example, CHO (chinese hamster ovarian) cell,BHK (baby hamster kidney) cell, lymphocyte such as myeloma) with thegene encoding said human monoclonal antibody (gene encoding the heavychain or that encoding the light chain, or both genes) isolated fromsaid human monoclonal antibody-producing B cell or human monoclonalantibody-producing fusion cell (hybridoma).

[0806] Herein, the genetic recombinant human monoclonalantibody-producing genetic recombinant cell aforementioned in (3) namelymeans a genetic recombinant cell producing the genetic recombinant ofhuman monoclonal antibody generated by the B cell described above in (1)or the hybridoma aforementioned in (2).

[0807] And, “host” in “genetic recombinant host” of this inventionincludes, in addition to various mammalian cells as described above,fertilized eggs of any non-human mammals (goat, swine, sheep, cattle,etc.). By transferring a gene (gene encoding the heavy chain or thatencoding the light chain, or both genes) encoding any monoclonalantibody (preferably human monoclonal antibody) to human AILIM of thisinvention into this fertilized egg, a genetic recombinant fertilized eggof this invention can be obtained. This genetic recombinant fertilizedegg is used to prepare transgenic animals for manufacturing theaforementioned protein from the milk on a large scale (Nikkei Science,April, 1997, p.78-84).

[0808] “A substance” composing the present invention, specifically “asubstance having an activity in modulating the signal transductionmediated by AILIM”, and more specifically “a substance having anactivity in inhibiting proliferation of AILIM-expressing cells, or ininhibiting production of a cytokine by AILIM-expressing cells” means anatural substance present in the nature, or a artificially preparedarbitrary substance.

[0809] “Substance” related to “substance binding to AILIM” and“substance binding to AILIM ligand” herein also means any naturalsubstance in nature or any artificially prepared substance.

[0810] Here, “the signal transduction mediated by AILIM” means thesignal transduction through AILIM, leading to a change of an arbitraryphenotype in the AILIM-expressing cells (cell proliferation, activationof cells, inactivation of cells, apoptosis, and/or a change of anability for producing an arbitrary cytokine from AILIM-expressingcells).

[0811] “The substance” can be mainly classified into “a proteinsubstance” and “a non-protein substance”.

[0812] Examples of the “protein substances” are the followingpolypeptide, antibody (a polyclonal antibody, a monoclonal antibody, ora portion of a monoclonal antibody, and particularly preferably thehuman antibody mentioned above).

[0813] When the substance is an antibody, the substance is preferably amonoclonal antibody. When the substance is a monoclonal antibody, thesubstance includes not only a non-human mammal derived monoclonalantibody, but also a recombinant chimeric monoclonal antibody, arecombinant humanized monoclonal antibody and human monoclonal antibody.

[0814] Here, the “recombinant chimeric monoclonal antibody” is amonoclonal antibody prepared by genetic engineering, and specificallymeans a chimeric antibody such as mouse/human chimeric monoclonalantibody whose variable regions are derived from immunoglobulin of annon-human mammal (mouse, rat, hamster, etc.) and whose constant regionsare derived from human immunoglobulin.

[0815] The “humanized monoclonal antibody (CDR-grafted antibody)” of thepresent invention is a monoclonal antibody prepared by geneticengineering and specifically means a humanized monoclonal antibodywherein a portion or the whole of the complementarity determiningregions of the hypervariable region are derived from the complementaritydetermining regions of the hypervariable region from a monoclonalantibody of an non-human mammal (mouse, rat, hamster, etc.), theframework regions of the variable region are derived from the frameworkregions of the variable region from human immunoglobulin, and theconstant region is derived from human a constant region fromimmnunoglobulin.

[0816] The complementarity determining regions of the hypervariableregion exists in the hypervariable region in the variable region of anantibody and means three regions which directly and complementary bindsto an antigen (complementarity-determining residues, CDR1, CDR2, andCDR3). The framework regions of the variable region mean fourcomparatively conserved regions lying upstream, downstream or betweenthe three complementarity determining regions (framework region, FR1,FR2, FR3, and FR4).

[0817] In other words, a humanized monoclonal antibody means that inwhich all the regions except a portion or the whole of thecomplementarity determining regions of the hypervariable region of anon-human mammal-derived monoclonal antibody have been replaced withtheir corresponding regions derived from a human immunoglobulin.

[0818] The constant region derived from human immunoglobulin has theamino acid sequence inherent in each isotype such as IgG (IgG1, IgG2,IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of a humanizedmonoclonal antibody in the present invention can be that from humanimmunoglobulin belonging to any isotype. Preferably, it is the constantregion of human IgG. The framework regions of the constant regionderived from human immunoglobulin are not particularly limited.

[0819] When the substance of the present invention is a polypeptide, thesubstance includes the following polypeptide, a fragment of thepolypeptide (an oligopeptide), a fusion polypeptide, a chemicallymodified one thereof. Examples of an oligopeptide are a peptidecomprising 5 to 30 amino acids, preferably 5 to 20 amino acids. Thechemical modification can be designed depending on various purposes, forexample, the increased half-life in blood in the case of administeringin vivo, or the increased tolerance against the degradation or increasedabsorption in digestive tract at the oral administration.

[0820] Examples of the polypeptide are as follows:

[0821] (1) A polypeptide comprising the whole or a portion of anextracellular region of AILIM;

[0822] (2) A fusion polypeptide comprising the whole or a portion of anextracellular region of AILIM and the whole or a portion of a constantregion of immunoglobulin heavy chain; or

[0823] (3) A polypeptide which binds to AILIM.

[0824] Examples of the “non-protein” are DNA, RNA, and a chemicallysynthesized compound.

[0825] Here, “DNA” means “DNA comprising a partial nucleotide sequenceof the DNA or chemically modified DNA thereof” useful as an antisenseDNA pharmaceutical designed based on a nucleotide sequence of DNA(including cDNA and genomic DNA) encoding the above AILIM (preferablyhuman AILIM). Specifically the antisense DNA can inhibit transcriptionof DNA encoding the AILIM into mRNA, or translation of the mRNA into aprotein by hybridizing DNA or RNA encoding AILIM.

[0826] The “partial nucleotide sequence” as referred to here indicates apartial nucleotide sequence comprising an arbitrary number ofnucleotides in an arbitrary region. The partial nucleotide sequenceconsists of 5 to 100 consecutive nucleotides, preferably 5 to 70consecutive nucleotides, more preferably 5 to 50 consecutivenucleotides, and still more preferably 5 to 30 consecutive nucleotides.

[0827] When the DNA is used as an antisense DNA pharmaceutical, the DNAsequence can be modified chemically in part for extending the half-life(stability) of the blood concentration of the DNA administered topatients, for increasing the intracytoplasmic-membrane permeability ofthe DNA, or for increasing the degradation resistance or the absorptionof the orally administered DNA in the digestive organs. The chemicalmodification includes, for example, the modification of the phosphatebonds, the riboses, the nucleotide bases, the sugar moiety, the 3′ endand/or the 5′ end in the structure of the oligonucleotide DNA.

[0828] The modification of phosphate bond includes, for example, theconversion of one or more of the bonds to phosphodiester bonds(D-oligo), phosphorothioate bonds, phosphorodithioate bonds (S-oligo),methyl phosphonate (MP-oligo), phosphoroamidate bonds, non-phosphatebonds or methyl phosphonothioate bonds, or combinations thereof. Themodification of the ribose includes, for example, the conversion to2′-fluororibose or 2′-O-methylribose. The modification of the nucleotidebase includes, for example, the conversion to 5-propynyluracil or2-aminoadenine.

[0829] Here, “RNA” means “RNA comprising a partial nucleotide sequenceof the RNA or chemically modified RNA thereof” useful as an antisenseRNA pharmaceutical designed based on a nucleotide sequence of RNAencoding the above AILIM (preferably human AILIM). The antisense RNA caninhibit transcription of DNA encoding the AILIM into mRNA, ortranslation of the mRNA into a protein by hybridizing DNA or RNAencoding AILIM.

[0830] The “partial nucleotide sequence” as referred to here indicates apartial nucleotide sequence comprising an arbitrary number ofnucleotides in an arbitrary region. The partial nucleotide sequenceconsists of 5 to 100 consecutive nucleotides, preferably 5 to 70consecutive nucleotides, more preferably 5 to 50 consecutivenucleotides, and still more preferably 5 to 30 consecutive nucleotides.

[0831] The sequence of antisense RNA can be modified chemically in partfor extending the half-life (stability) of the blood concentration ofthe RNA administered to patients, for increasing theintracytoplasmic-membrane permeability of the RNA, or for increasing thedegradation resistance or the absorption of the orally administered RNAin the digestive organ. An example of chemical modification is thechemical modification applied to the above antisense DNA.

[0832] Examples of “a chemically synthesized compound” are an arbitrarycompound except for the above DNA, RNA and protein substances, havingthe molecular weight of about 100 to about 1000, preferably a compoundhaving the molecular weight of about 100 to about 800, and morepreferably the molecular weight of about 100 to about 600.

[0833] A “polypeptide” included in the definition of the above“substance” means a portion (a fragment) of a polypeptide chainconstituting AILIM (preferably human AILIM), preferably the whole or aportion of an extracellular region of the polypeptide constituting AILIM(1 to 5 amino acids may be optionally added into the N-terminus and/orC-terminus of the region).

[0834] AILIM involving in the present invention is a transmembranemolecule penetrating cell membrane, comprising 1 or 2 polypeptidechains.

[0835] Here, a “transmembrane protein” means a protein that connectswith membrane through the hydrophobic peptide region penetrating thelipid bilayer of the membrane once or several times and whose structureis, as a whole, composed of three main regions, that is, extracellularregion, transmembrane region, and cytoplasmic region, as seen in manyreceptors or cell surface molecules. Such a transmembrane proteinconstitutes each receptor or cell surface molecule in the form of amonomer, homodimer, heterodimer or oligomer with another chain(s) havingthe same or different amino acid sequence.

[0836] Here, an “extracellular region” means the whole or a portion fromthe partial structure (partial region) from the entire structure of theabove-mentioned transmembrane protein where the partial structure existsoutside of the membrane. In other words, it means the whole or a portionof the region of the transmembrane protein except the regionincorporated into the membrane (transmembrane region) and the regionexisting in the cytoplasm following the transmembrane region(cytoplasmic region).

[0837] “A fusion polypeptide” included in the above “protein substance”means a fusion polypeptide comprising the whole or a portion of anextracellular region of a polypeptide constituting AILIM (preferablyhuman AILIM), and “the whole or a portion of a constant region ofimmunoglobulin heavy chain (Ig, preferably human Ig)”. Preferably, thefusion polypeptide is a fusion polypeptide with an extracellular regionof AILIM and a portion of a constant region of human IgG heavy chain andparticularly preferably, a fusion polypeptide of an extracellular regionof AILIM and a region (Fc) of human IgG heavy chain comprising a hingeregion, CH2 domain and CH3 domain. As IgG, IgG1 is preferable, and asAILIM, human, mouse, or rat AILIM is preferable (preferably human).

[0838] “The whole or a portion of a constant region of humanimmunoglobulin (Ig) heavy chain” used herein means the constant regionor the Fc region of human-derived immunoglobulin heavy chain (H chain)as described, or a portion thereof. The immunoglobulin can be anyimmunoglobulin belonging to any class and any subclass. Specifically,examples of the immunoglobulin are IgG (IgG1, IgG2, IgG3, and IgG4),IgM, IgA (IgA1 and IgA2), IgD, and IgE. Preferably, the immunoglobulinis IgG (IgG1, IgG2, IgG3, or IgG4), or IgM. Examples of particularlypreferable immunoglobulin of the present invention are those belongingto human-derived IgG (IgG1, IgG2, IgG3, or IgG4).

[0839] Immunoglobulin has a Y-shaped structural unit in which fourchains composed of two homologous light chains (L chains) and twohomologous heavy chains (H chains) are connected through disulfide bonds(S-S bonds). The light chain is composed of the light chain variableregion (V_(L)) and the light chain constant region (C_(L)). The heavychain is composed of the heavy chain variable region (V_(H)) and theheavy chain constant region (C_(H)).

[0840] The heavy chain constant region is composed of some domainshaving the amino acid sequences inherent in each class (IgG, IgM, IgA,IgD, and IgE) and each subclass (IgG1, IgG2, IgG3, and IgG4, IgA1, andIgA2).

[0841] The heavy chain of IgG (IgG1, IgG2, IgG3, and IgG4) is composedof VH, CH1 domain, hinge region, CH2 domain, and CH3 domain in thisorder from N terminus.

[0842] Similarly, the heavy chain of IgG1 is composed of V_(H), Cγ₁1domain, hinge region, Cγ₁2 domain, and Cγ₁3 domain in this order from Nterminus. The heavy chain of IgG2 is composed of V_(H), Cγ₂1 domain,hinge region, Cγ₂2 domain, and Cγ₂3 domain in this order from Nterminus. The heavy chain of IgG3 is composed of V_(H), Cγ₃1 domain,hinge region, Cγ₃2 domain, and Cγ₃3 domain in this order from Nterminus. The heavy chain of IgG4 is composed of V_(H), Cγ₄1 domain,hinge region, Cγ₄2 domain, and Cγ₄3 domain in this order from Nterminus.

[0843] The heavy chain of IgA is composed of V_(H), Cα1 domain, hingeregion, Cα2 domain, and Cα3 domain in this order from N terminus.

[0844] Similarly, the heavy chain of IgA1 is composed of V_(H), Cα₁1domain, hinge region, Cα₁2 domain, and Cα₁3 domain in this order from Nterminus. The heavy chain of IgA2 is composed of V_(H), Cα₂1 domain,hinge region, Cα₂2 domain, and Cα₂3 domain in this order from Nterminus.

[0845] The heavy chain of IgD is composed of V_(H), Cδ1 domain, hingeregion, Cδ2 domain, and Cδ3 domain in this order from N terminus.

[0846] The heavy chain of IgM is composed of V_(H), Cμ1 domain, Cμ2domain, Cμ3 domain, and Cμ4 domain in this order from N terminus andhave no hinge region as seen in IgG, IgA, and IgD.

[0847] The heavy chain of IgE is composed of V_(H), Cε1 domain, Cε2domain, Cε3 domain, and Cε4 domain in this order from N terminus andhave no hinge region as seen in IgG, IgA, and IgD.

[0848] If, for example, IgG is treated with papain, it is cleaved at theslightly N terminal side beyond the disulfide bonds existing in thehinge region where the disulfide bonds connect the two heavy chains togenerate two homologous Fab, in which a heavy chain fragment composed ofV_(H) and CH1 is connected with one light chain through a disulfidebond, and one Fc, in which two homologous heavy chain fragments composedof the hinge region, CH2 domain, and CH3 domain are connected throughdisulfide bonds (See “Immunology Illustrated”, original 2nd ed.,Nankodo, pp.65-75 (1992); and “Focus of Newest Medical Science‘Recognition Mechanism of Immune System’”, Nankodo, pp.4-7(1991); and soon).

[0849] Namely, “a portion of a constant region of immunoglobulin heavychain” mentioned above means a portion of a constant region of animmunoglobulin heavy chain having the structural characteristics asmentioned above, and preferably, is the constant region without C1domain, or the Fc region. Specifically, example thereof is the regioncomposed of hinge region, C2 domain, and C3 domain from each of IgG,IgA, and IgD, and is the region composed of C2 domain, C3 domain, and C4domain from each of IgM and IgE. A particularly preferable examplethereof is the Fc region of human-derived IgG1.

[0850] The fusion polypeptide mentioned above has the advantage that thefusion polypeptide can be purified extremely easily by using affinitycolumn chromatography using the property of protein A, which bindsspecifically to the immunoglobulin fragment because the fusionpolypeptide of the present invention has a portion of a constant region(for example Fc) of an immunoglobulin such as IgG as mentioned above asa fusion partner. Moreover, since various antibodies against the Fc ofvarious immunoglobulins are available, an immunoassay for the fusionpolypeptides can be easily performed with antibodies against the Fc.

[0851] “A polypeptide which binds to AILIM” is included in “apolypeptide” included in the definition of the above “substance”.

[0852] Specific examples of “a polypeptide which binds to AILIM” are thewhole or a portion of a polypeptide constituting known B7h, B7RP-1, GL50or a molecule called LICOS which are ligands interacting with AILIM(Nature, Vol.402, No.6763, pp.827-832, 1999; Nature Medicine, Vol.5,No.12, pp.1365-1369, 1999; J. Immunology, Vol.164, pp.1653-1657, 2000;Curr. Biol., Vol.10 No 6, pp.333-336, 2000).

[0853] Preferably, the polypeptide is a polypeptide comprising the wholeor a portion of an extracellular region of the above ligands (B7h,B7RP-1, GL50, LICOS), or a fusion polypeptide comprising thepolypeptide, and the whole or a portion of a constant region ofimmunoglobulin heavy chain (preferably human immunoglobulin). Here, theterms “an extracellular region” and “a constant region of immunoglobulinheavy chain” have the same meaning as the above.

[0854] The polypeptide, a portion of the polypeptide (fragment), andfusion polypeptide mentioned above can be produced not only byrecombinant DNA technology as mentioned below but also by a method wellknown in the art such as a chemical synthetic method and a cell culturemethod, or a modified method thereof.

[0855] The “antibody” of the present invention can be a polyclonalantibody (antiserum) or a monoclonal antibody against mammalian AILIM(particularly preferably human AILIM) defined above, and preferably amonoclonal antibody.

[0856] Specifically the antibody is an antibody having an activity ininhibiting proliferation of AILIM-expressing cells by biding to AILIM,or inhibiting production of interferon γ or interleukin 4 byAILIM-expressing cells through biding to AILIM.

[0857] “Delayed type allergy” herein this allergy mediated by cellularimmunity (particularly mediated by Th1-type T cell), that is, theallergy is mediated by T cell sensitized with antigen (memory T cellmemorizing antigen) and is referred to any allergy, which takesapproximately 24 to 48 hours to exhibit allergic reaction accompaniedwith inflammation caused by said memory T cell when the living organismsensitized with an antigen is re-contacted with the same antigen.

[0858] This delayed type allergy includes allergy to an infectiouspathogenic antigen such as tuberculin allergy derived from Mycobacteriumtuberculosis, a transient Jones-Mote delayed type allergy to a minutequantity of protein, contact allergy to chemicals such as picrylchloride or plant toxin such as lacquer, or allergy related to graftrejection to graft observed in the allograft.

[0859] “Pharmaceutical composition” herein means a composition useful asa drug comprising as the effective ingredients antibody (preferablyhuman antibody), which binds to AILIM (preferably human AILIM) or aportion thereof, or monoclonal antibody (preferably human monoclonalantibody) or a portion thereof and a “pharmacologically acceptablecarrier”.

[0860] The “pharmaceutically acceptable carrier” includes a excipient, adiluent, an expander, a decomposition agent, a stabilizer, apreservative, a buffer, an emulsifier, an aromatic, a colorant, asweetener, a viscosity increasing agent, a flavor, a solubilityincreasing agent, or other additives.

[0861] Using one or more of such carriers, a pharmaceutical compositioncan be formulated into tablets, pills, powders, granules, injections,solutions, capsules, troches, elixirs, suspensions, emulsions, orsyrups.

[0862] The pharmaceutical composition can be administered orally orparenterally. Other forms for parenteral administration include asolution for external application, suppository for rectaladministration, and pessary, prescribed by the usual method, whichcomprises one or more active ingredient.

[0863] The dosage can vary depending on the age, sex, weight, andsymptom of a patient, effect of treatment, administration route, periodof treatment, or the kind of active ingredient (polypeptide or antibodymentioned above) contained in the pharmaceutical composition. Usually,the pharmaceutical composition can be administered to an adult in a doseof 10 μg to 1000 mg (or 10 μg to 500 mg) per one administration.Depending on various conditions, the dosage less than that mentionedabove may be sufficient in some cases, and the dosage more than thatmentioned above may be necessary in other cases.

[0864] In particular, the injection can be produced by dissolving orsuspending the antibody in a non-toxic, pharmaceutically acceptablecarrier such as physiological saline or commercially available distilledwater for injection with adjusting a concentration to 0.1 μg antibody/mlcarrier to 10 mg antibody/ml carrier.

[0865] The injection thus produced can be administered to a humanpatient in need of treatment in a dose of 1 μg to 100 mg/kg body weight,preferably 50 μg to 50 mg/kg body weight once or more times a day.Examples of administration route are medically appropriateadministration routes such as intravenous injection, subcutaneousinjection, intradermal injection, intramuscular injection, orintraperitoneal injection, preferably intravenous injection.

[0866] The injection can also be prepared into a non-aqueous diluent(for example, propylene glycol, polyethylene glycol, vegetable oil suchas olive oil, and alcohol such as ethanol), suspension, or emulsion.

[0867] The injection can be sterilized by filtration with abacteria-non-penetrated filter, by mixing bacteriocide, or byirradiation. The injection can be produced in the form that is preparedupon use. Namely, it is freeze-dried to be a sterile solid composition,and can be dissolved in sterile distilled water for injection or anothersolvent before use.

[0868] Pharmaceutical compositions comprising the human antibodies ofthis invention are useful as pharmaceutical preparations, withoutinducing host immunorejection due to HAMA (human anti-mouse antibody),to control a variety of biological reactions (e.g., proliferation ofcells expressing AILIM, cytokine production by cells expressing AILIM,immune cytolysis or death (apoptosis) of cells expressing AILIM andothers) that are associated with AILIM-mediated transduction ofcostimulatory signal (secondary signal) to AILIM-expressing cells,and/or as pharmaceutical preparations to treat or prevent variousdiseases by suppressing and inhibiting the onset and/or progress ofdiseases associated with AILIM-mediated signal transduction.

[0869] The term “immune cytolysis” herein indicates a biologicalphenomenon as follows.

[0870] Lysis of the cell (cytolysis) can be induced by an antibody(particularly cell-lysing antibody) as well as by binding with killercells. The cell-lysing antibody is a cytotoxic antibody, whichparticularly has lysing activity on cells such as immune cells, tissuecell or sperms. When the antibody binds to the cell-surface antigen, itcauses a cytotoxic effect on the cell or induces cytolysis in thepresence of the complement.

[0871] This immune cytolysis is induced by the action of the complementin conjunction with specific binding of the antibody to cell-surfaceantigen. The antibody bound to the surface antigen activates C1complement (C1). Subsequently cell damage sites are formed through aseries of complement-fixation reactions with C2 to C9 complements(C2-C9), and then cellular contents are released from the cells therebylysing the cells.

[0872] The term “antibody-dependent cellular cytotoxicity” hereinindicates a biological event that is also abbreviated as “ADCC,” and isa cytotoxic action on target cells by effector cells such aslymphocytes, macrophages or polymorphonuclear leucocytes, which requiresnot only effector cells and target cells but also an antibodyparticipating in the induction of the cytotoxic event.

[0873] The term “mixed lymphocyte reaction” herein means a biologicalphenomenon abbreviated as “MLR.” The reaction is also referred to asmixed leukocyte reaction.

[0874] Allogenic leukocytes or lymphocytes derived from distinctindividuals are mixed with each other and cultured for several days,thereby allowing blast formation of the cells and DNA synthesis in thecells (i.e., cell proliferation). This reaction is referred to as MLR(allogenic MLR).

[0875] DNA synthesis (cell proliferation) can be analyzed by arrestingproliferation of either of the lymphocytes. The arrest can beaccomplished by treatment with irradiation or mitomycin. Analysis can becarried out by measuring the amount of DNA synthesized in the otherlymphocyte.

[0876] The amount of synthesized DNA can be analyzed by measuringincorporation of thymidine, labeled with radioisotope such as tritium,into the nucleus of the cell.

[0877] DNA encoding AILIM (particularly preferably human AILIM) of thepresent invention can be obtained according to a commonly used method byusing procedures for cloning cDNA from mRNA encoding AILIM; procedurefor isolating genomic DNA and splicing them; procedure for preparing theDNA by PCR using a cDNA sequence or mRNA sequence as a template; orprocedure for chemically synthesizing the DNA.

[0878] DNA encoding the AILIM ligand in accordance with the presentinvention can also be obtained in the same manner as described above.

[0879] DNA encoding AILIM (particularly preferably the human AILIM) ofthe present invention can be prepared by cutting (digesting) DNAcomprising DNA encoding AILIM prepared as such with appropriaterestriction enzymes, and as required, ligating the resultant DNAfragment with a linker DNA or tag by using an appropriate DNA polymeraseor the like. The DNA encoding AILIM ligand can also be prepared in thesame manner.

[0880] An exemplary method will be shown below to clone the cDNAencoding AILIM (particularly preferably the human AILIM; the protein ishereinafter referred to as the protein of interest) from the mRNA.

[0881] A DNA encoding AILIM ligand can also be cloned in the samemanner.

[0882] First, messenger RNA encoding the protein of interest is preparedfrom tissues or cells expressing and producing the protein of interest.mRNA can be prepared isolating total RNA by a known method such asquanidine-thiocyanate method (Chirgwin, J. M. et al., Biochemistry,Vol.18, p5294, 1979), hot phenol method, or AGPC method, and subjectingit to affinity chromatography using oligo-dT cellulose or poly-USepharose.

[0883] Then, with the mRNA obtained as a template, cDNA is synthesized,for example, by a well-known method using reverse transcriptase such asthe method of Okayama et al. (Mol. Cell. Biol. Vol.2, p.161 (1982);ibid. Vol.3, p.280 (1983)) or the method of Hoffman et al. (Gene Vol.25,p.263 (1983)), and converted into double-stranded cDNA. A cDNA libraryis prepared by transforming E. coli with plasmid vectors, phage vectors,or cosmid vectors having this cDNA or by transfecting E. coli after invitro packaging.

[0884] The plasmid vectors used in this invention are not limited aslong as they are replicated and maintained in hosts. Any phage vectorsthat can be replicated in hosts can also be used. Examples of usuallyused cloning vectors are pUC19, λgt10, λgt11, and so on. When the vectoris applied to immunological screening as mentioned below, the vectorhaving a promoter that can express a gene encoding the polypeptide ofthe present invention in a host is preferably used.

[0885] cDNA can be inserted into a plasmid by, for example, the methodof Maniatis et al. (Molecular Cloning, A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA can beinserted into a phage vector by, for example, the method of Hyunh et al.(DNA cloning, a practical approach, Vol. 1, p.49 (1985)). These methodscan be simply performed by using a commercially available cloning kit(for example, a product from Takara Shuzo). The recombinant plasmid orphage vector thus obtained is introduced into appropriate host cellssuch as a prokaryote (for example, E. coli: XL1Blue MRF′, DH5α, HB101,MC1061/P3, etc.).

[0886] Examples of a method for introducing a plasmid into a host arecalcium chloride method, calcium chloride/rubidium chloride methoddescribed in Molecular Cloning, A Laboratory Manual (second edition,Cold Spring Harbor Laboratory, p.1.74 (1989)), and electroporationmethod. Phage vectors can be introduced into host cells by, for example,a method in which the phage DNAs are introduced into grown hosts afterin vitro packaging. In vitro packaging can be easily performed with acommercially available in vitro packaging kit (for example, a productfrom Stratagene or Amersham).

[0887] The cDNA encoding the polypeptide of the present invention can beisolated from the cDNA library so prepared according to the methodmentioned above by combining general cDNA screening methods.

[0888] For example, a clone comprising the desired cDNA can be screenedby a known colony hybridization method (Crunstein et al. Proc. Natl.Acad. Sci. USA, Vol.72, p.3961 (1975)) or plaque hybridization method(Molecular Cloning, A Laboratory Manual, second edition, Cold SpringHarbor Laboratory, p.2.108 (1989)) using ³²P-labeled chemicallysynthesized oligonucleotides as probes, which are corresponding to theamino acid sequence of the polypeptide of the present invention.Alternatively, a clone having a DNA fragment encoding a specific regionwithin the polypeptide of the present invention can be screened byamplifying the region by PCR with synthetic PCR primers.

[0889] When a cDNA library prepared using a cDNA expression vector (forexample, λZAPII phage vector) is used, the desired clone can be screenedby the antigen-antibody reaction using an antibody against thepolypeptide of the present invention. A screening method using PCRmethod is preferably used when many clones are subjected to screening.

[0890] The nucleotide sequence of the DNA thus obtained can bedetermined by Maxam-Gilbert method (Maxam et al. Proc. Natl. Acad. Sci.USA, Vol.74, p.560 (1977)) or the dideoxynucleotide synthetic chaintermination method using phage M13 (Sanger et al. Proc. Natl. Acad. Sci.USA, Vol.74, pp.5463-5467 (1977)). The whole or a portion of the geneencoding the polypeptide of the present invention can be obtained byexcising the clone obtained as mentioned above with restriction enzymesand so on.

[0891] The DNA encoding the polypeptide of the present invention can beisolated from the genomic DNA derived from the cells expressing thepolypeptide of the present invention as mentioned above by the followingmethods.

[0892] Such cells are solubilized preferably by SDS or proteinase K, andthe DNAs are deproteinized by repeating phenol extraction. RNAs aredigested preferably with ribonuclease. The DNAs obtained are partiallydigested with appropriate restriction enzymes, and the DNA fragmentsobtained are amplified with appropriate phage or cosmid to generate alibrary. Then, clones having the desired sequence are detected, forexample, by using radioactively labeled DNA probes, and the whole or aportion of the gene encoding the polypeptide of the present invention isobtained from the clones by excision with restriction enzyme and so on.

[0893] Preparation of DNA encoding the protein of interest by PCR can becarried out by using known mRNA or cDNA encoding the protein of interestas the template according to a usual method (“PCR techniques for geneamplification—fundamental and new technologies” KYORITSU SHUPPAN, 1992,etc.).

[0894] The DNA encoding the protein of interest can also be chemicallysynthesized by the usual method, based on the nucleotide sequenceencoding the protein of interest.

[0895] AILIM of the invention(particularly preferably the human AILIM)or a portion thereof (preferably, the extracellular region) can beprepared as a recombinant protein according to a usual method withcommonly used genetic recombination techniques, using DNA obtained bycutting DNA encoding AILIM (cDNA or intron-containing genomic DNA) basedon the method illustrated above with appropriate restriction enzymes togive a DNA fragment encoding the AILIM, and then as required, ligatingthe resultant DNA fragment with a linker DNA or tag, by using anappropriate DNA polymerase or the like.

[0896] The AILIM ligand (particularly preferably the human AILIM ligand)or a portion thereof (preferably, the extracellular region) can beprepared in the same manner.

[0897] A specific example is illustrated below. Namely, the DNA preparedas described above is inserted into a vector, which will be describedlater in detail, to yield an expression vector. Then the expressionvector is used to transform a host cell as described below to obtain atransformant. The transformant is cultured and allowed to produce theprotein of interest into the culture supernatant. The protein ofinterest in the culture supernatant can easily be purified by usingcolumn chromatography and such.

[0898] There is no particular limitation on the type of expressionvector for the production of the recombinant AILIM (or its extracellularregion), as far as the vector is replicated and maintained or producedautonomously in any of various hosts such as prokaryotic cells and/oreukaryotic cells. Such expression vectors include plasmid vectors andphage vectors (Cloning Vectors: A Laboratory Manual, Elsevier, New York,1985).

[0899] The expression vector can easily be prepared by ligating the DNAencoding AILIM (or its extracellular region) with a vector forrecombination available in the art (plasmid DNA and bacteriophage DNA)by the usual method. Specific examples of the vectors for recombinationused are E. coli-derived plasmids such as pBR322, pBR325, pUC12, pUC13,and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillussubtilis-derived plasmids such as pUB110, pTP5, and pC194. Examples ofphages are a bacteriophage such as λ phage, and an animal or insectvirus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, andnuclear polyhedrosis virus.

[0900] Plasmid vectors are useful, when a DNA encoding AILIM of theinvention (particularly preferably the human AILIM) or its solubleextracellular region is intended to be expressed in a host cell andthereby expressing the AILIM on the surface of the host cell, oralternatively the soluble extracellular region of the AILIM(particularly preferable the human AILIM) is intended to be produced.There is no particular limitation on such plasmid vectors, as far as thevectors can express the gene encoding AILIM (particularly preferably thehuman AILIM) or its soluble extracellular region and produce the encodedprotein in various host cells such as prokaryotic cells and/oreukaryotic cells. For example, such plasmids include pMAL C2, pcDNA3.1(−), pEF-BOS (Nucleic Acid Research, Vol.18, p.5322, 1990; etc.), pME18S(“Handbook for genetic engineering,” Experimental Medicine, supplement,1992; etc.), etc.

[0901] When bacteria, particularly E. coli are used as host cells, anexpression vector is generally comprised of, at least, apromoter-operator region, an initiation codon, the DNA encoding theprotein of the present invention, termination codon, terminator region,and replicon.

[0902] When yeast, animal cells, or insect cells are used as hosts, anexpression vector is preferably comprised of, at least, a promoter, aninitiation codon, the DNA encoding the AILIM (particularly preferablyhuman AILIM) of the present invention or its extracellular region, and atermination codon. It may also comprise the DNA encoding a signalpeptide, enhancer sequence, 5′- and 3′-untranslated region of the geneencoding the AILIM of the present invention, splicing junctions,polyadenylation site, selectable marker region, and replicon. Theexpression vector may also contain, if required, a gene for geneamplification (marker) that is usually used.

[0903] A promoter-operator region to express the AILIM (particularlypreferably human AILIM) of the present invention or its extracellularregion in bacteria comprises a promoter, an operator, and aShine-Dalgarno (SD) sequence (for example, AAGG). For example, when thehost is Escherichia, it preferably comprises Trp promoter, lac promoter,recA promoter, λPL promoter, lpp promoter, tac promoter, or the like.

[0904] Examples of a promoter to express the AILIM (particularlypreferably human AILIM) of the present invention or its extracellularregion in yeast are PH05 promoter, PGK promoter, GAP promoter, ADHpromoter, and so on. When the host is Bacillus, examples thereof areSL01 promoter, SP02 promoter, penP promoter and so on.

[0905] When the host is a eukaryotic cell such as a mammalian cell,examples thereof are SV40-derived promoter, retrovirus promoter, heatshock promoter, and so on. As a matter of course, the promoter is notlimited to the above examples. In addition, to use an enhancer iseffective for expression.

[0906] A preferable initiation codon is, for example, a methionine codon(ATG).

[0907] The commonly used termination codon (for example, TAG, TGA, TAA,and so on) is illustrated as a termination codon.

[0908] Usually used natural or synthetic terminators are used as aterminator region.

[0909] A replicon means a DNA capable of replicating the whole DNAsequence in host cells, and includes a natural plasmid, an artificiallymodified plasmid (DNA fragment prepared from a natural plasmid), asynthetic plasmid, and so on. Examples of a preferable plasmids arepBR322 or its artificial derivatives (DNA fragment obtained by treatingpBR322 with appropriate restriction enzymes) for E. coli, yeast 2μplasmid or yeast chromosomal DNA for yeast, and pRSVneo ATCC 37198,pSV2dhfr ATCC 37145, pdBPV-MMTneo ATCC 37224, pSV2neo ATCC 37149,pSV2bsr, etc. for mammalian cells.

[0910] An enhancer sequence, polyadenylation site, and splicing junctionthat are usually used in the art, such as those derived from SV40 can bealso used.

[0911] A selectable marker usually used can be used according to theusual method. Examples thereof are resistance genes for antibiotics,such as tetracycline, ampicillin, or kanamycin, and thymidine kinasegene.

[0912] Examples of a gene for gene amplification are dihydrofolatereductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene,glutamate synthase gene, adenosine deaminase gene, ornithinedecarboxylase gene, hygromycin-B-phophotransferase gene, aspartatetranscarbamylase gene, etc.

[0913] The expression vector of the present invention can be prepared bycontinuously and circularly linking at least the above-mentionedpromoter, initiation codon, DNA encoding the protein of the presentinvention, termination codon, and terminator region, to an appropriatereplicon. If desired, appropriate DNA fragments (for example, linkers,restriction sites generated with other restriction enzyme), can be usedby the usual method such as digestion with a restriction enzyme orligation using T4 DNA ligase.

[0914] Transformants of the present invention can be prepared byintroducing the expression vector mentioned above into host cells.

[0915] Host cells used in the present invention are not limited as longas they are compatible with an expression vector mentioned above and canbe transformed. Examples thereof are various cells such as natural cellsor artificially established recombinant cells usually used in technicalfield of the present invention (for example, bacteria (Escherichia andBacillus), yeast (Saccharomyces, Pichia, etc.), animal cells, or insectcells.

[0916]E. coli or animal cells are preferably used. Specific examples areE. coli (DH5α, DH10B, TB1, HB101, XL-2Blue, etc.), mouse-derived cells(COP, L, C127, Sp2/0, NS-1, NIH 3T3, etc.), rat-derived cells,hamster-derived cells (BHK, CHO, etc.), monkey-derived cells (COS1,COS3, COS7, CV1, Velo, etc.), and human-derived cells (Hela, diploidfibroblast-derived cells, myeloma, Namalwa, etc.).

[0917] An expression vector can be introduced (transformed (transduced))into host cells by known method.

[0918] Transformation can be performed, for example, according to themethod of Cohen et al. (Proc. Natl. Acad. Sci. USA, Vol.69, p.2110(1972)), protoplast method (Mol. Gen. Genet., Vol.168, p.111 (1979)), orcompetent method (J. Mol. Biol., Vol.56, p.209 (1971)) when the hostsare bacteria (E. coli, Bacillus subtilis, etc.), the method of Hinnen etal. (Proc. Natl. Acad. Sci. USA, Vol.75, p.1927 (1978)), or lithiummethod (J. Bacteriol., Vol.153, p.163 (1983)) when the host isSaccharomyces cerevisiae, the method of Graham (Virology, Vol.52, p.456(1973)) when the hosts are animal cells, and the method of Summers etal. (Mol. Cell. Biol., Vol.3, pp.2156-2165 (1983)) when the hosts areinsect cells.

[0919] The extracellular region of the AILIM (particularly preferablyhuman AILIM) of the present invention (soluble AILIM) can be produced bycultivating transformants (in the following this term includestransductants) comprising an expression vector prepared as mentionedabove in nutrient media. AILIM ligand can be produced in the same way.

[0920] The nutrient media preferably comprise carbon source, inorganicnitrogen source, or organic nitrogen source necessary for the growth ofhost cells (transformants). Examples of the carbon source are glucose,dextran, soluble starch, and sucrose, and examples of the inorganic ororganic nitrogen source are ammonium salts, nitrates, amino acids, cornsteep liquor, peptone, casein, meet extract, soy bean cake, and potatoextract. If desired, they may comprise other nutrients (for example, aninorganic salt (for example, calcium chloride, sodiumdihydrogenphosphate, and magnesium chloride), vitamins, antibiotics (forexample, tetracycline, neomycin, ampicillin, kanamycin, etc.).

[0921] Cultivation is performed by a method known in the art.Cultivation conditions such as temperature, pH of the media, andcultivation time are selected appropriately so that the protein of thepresent invention is overproduced.

[0922] Specific media and cultivation conditions used depending on hostcells are illustrated below, but are not limited thereto.

[0923] When the hosts are bacteria, actinomycetes, yeasts, filamentousfungi, liquid media comprising the nutrient source mentioned above areappropriate. The media with pH 5 to 8 are preferably used.

[0924] When the host is E. coli, examples of preferable media are LBmedia, M9 media (Miller et al. Exp. Mol. Genet., Cold Spring HarborLaboratory, p.431 (1972)), YT media, etc. Using these media, cultivationcan be performed usually at 14 to 43° C. for about 3 to 24 hours withaeration and stirring, if necessary.

[0925] When the host is Bacillus, cultivation can be performed usuallyat 30 to 40° C. for about 16 to 96 hours with aeration and stirring, ifnecessary.

[0926] When the host is yeast, examples of media are Burkholder minimalmedia (Bostian, Proc. Natl. Acad. Sci. USA, Vol.77, p.4505 (1980)). ThepH of the media is preferably 5 to 8. Cultivation can be performedusually at 20 to 35° C. for about 14 to 144 hours with aeration andstirring, if necessary.

[0927] When the host is an animal cell, examples of media are MEM mediacontaining about 5 to 20% fetal bovine serum (Science, Vol.122, p.501(1952)), DMEM media (Virology, Vol.8, p.396 (1959)), RPMI1640 media (J.Am. Med. Assoc., Vol.199, p.519 (1967)), 199 media (Proc. Soc. Exp.Biol. Med., Vol.73, p.1 (1950)), HamF12 media, etc. The pH of the mediais preferably about 6 to 8. Cultivation can be performed usually atabout 30 to 40° C. for about 15 to 72 hours with aeration and stirring,if necessary.

[0928] When the host is an insect cell, an example of media is Grace'smedia containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol.82,p.8404 (1985)). The pH thereof is preferably about 5 to 8. Cultivationcan be performed usually at about 20 to 40° C. for 15 to 100 hours withaeration and stirring, if necessary.

[0929] The extracellular region (soluble AILIM) of AILIM of theinvention (particularly preferably the human AILIM) can be produced byculturing the above-mentioned transformed cells (particularly, animalcell or E. coli) and allowing the secretion of the protein in theculture supernatant. Namely, a culture filtrate (supernatant) isobtained by the method such as filtration or centrifugation of theobtained culture, and the polypeptide or polypeptide fragment of thepresent invention is purified and isolated from the culture filtrate bythe usual method commonly used in order to purify and isolate a naturalor synthetic protein.

[0930] Examples of the isolation and purification method are a methodutilizing specific affinity, such as affinity chromatography, a methodutilizing solubility, such as salting out and solvent precipitationmethod, a method utilizing the difference in molecular weight, such asdialysis, ultrafiltration, gel filtration, and sodium dodecylsulfate-polyacrylamide gel electrophoresis, a method utilizing charges,such as ion exchange chromatography and hydroxylapatite chromatography,a method utilizing the difference in hydrophobicity, such as reversephase high performance liquid chromatography, and a method utilizing thedifference in isoelectric point, such as isoelectric focusing.

[0931] When the protein of interest exists in the periplasm or cytoplasmof cultured transformants, first, the fungus bodies or cells areharvested by the usual method such as filtration or centrifugation andsuspended in appropriate buffer. After the cell wall and/or cellmembrane of the cells and so on are disrupted by the method such aslysis with sonication, lysozyme, and freeze-thawing, the membranefraction comprising the polypeptide of the present invention is obtainedby the method such as centrifugation or filtration. The membranefraction is solubilized with a detergent such as Triton-X100 to obtainthe crude extract. Finally, the polypeptide or the polypeptide fragmentis isolated and purified from the crude extract by the usual method asillustrated above.

[0932] In the present invention, the term “insoluble carrier” means acarrier which is used to immobilize polypeptides on them by physicaladsorption or chemical linking. For example, the carrier can be (1)plate, test tube, tube, or the like having internal space, bead, ball,filter, membrane, or the like made of water-insoluble materialsincluding plastics such as polystyrene resin, polycarbonate resin,silicon resin or nylon resin, or glass, and (2) insoluble carrier usedin affinity chromatography such as cellulose carrier, agarose carrier,polyacrylamide carrier, dextran carrier, polystyrene carrier,polyvnylalcohol carrier, poly amino acid carrier, porous silica carrier,etc.

[0933] The “labeling substance capable of giving detectable signal” inaccordance with the present invention includes, for example, enzyme,fluorescent material, luminescent material, biotin, avidin orradioisotope, more specifically, enzymes such as peroxidase (e.g.,horseradish peroxidase), alkaline phosphatase, β-D-galactosidase,glucoseoxidase, glucose-6-phosphate dehydrogenase, alcoholdehydrogenase, malate dehydrogenase, penicillinase, catalase,apo-glucose oxidase, urease, luciferase, acetylcholine esterase, etc.;fluorescent materials such as fluorescein isothiocyanate, phycobilinprotein, rare earth metal chelating agents, dansyl chloride, tetramethylrhodamine isothiocyanate, etc.; radioisotopes such as ³H, ¹⁴C, ¹²⁵I,¹³¹I, etc.; biotin, avidin, and luminescent material.

[0934] Among them, radioisotope or fluorescent material can givedetectable signal even when used alone. On the other hand, when usedalone, enzyme, luminescent material, biotin or avidin provide nodetectable signal, but when allowed to react with one or moresubstances, it can provide detectable signal. For example, when thelabel is an enzyme, at least a substrate is necessary for the detection.Various types of substrates can be used depending on the type of methodfor measuring enzyme activity (colorimetry, fluoroscopy, method usingbioluminescence or chemical luminescence, etc.). For example, when thelabel is peroxidase, hydrogen peroxide can be used as a substrate.Alternatively, when the label is biotin, avidin or enzyme-modifiedavidin is commonly used but is not limited to them. As required, avariety of luminescent substances can be utilized depending on the typeof substrate to be used.

[0935] Any of the above-mentioned labels can be utilized in the presentinvention. However, preferred label is an enzyme such as peroxidase orbiotin with consideration given to sensitivity of detection or assay aswell as the convenience of manipulation.

[0936] A “method for identifying a substance capable of binding to AILIMor AILIM ligand” in accordance with the invention is constructed basedon the principle of immunoassay.

[0937] Specifically, the principles of various methods as described in“Immunoassay (3^(rd) Edition, eds., Eiji Ishikawa et al, Igakushoin,1987)” can be applied.

[0938] Examples of principles preferably used include solid-phaseone-antibody method, liquid-phase two-antibody method, solid-phasetwo-antibody method, sandwich method, and one-pot method as described inExamined Published Japanese Patent Application (JP-B) No. Hei 2-39747.Further, assay method employing antigen-antibody reaction is exemplifiedby EMIT method (enzyme multiplied immunoassay technique), enzymechanneling immunoassay, enzyme modulator mediated enzyme immunoassay(EMMIA), enzyme inhibitor immunoassay, immunoenzymometric assay, enzymeenhanced immunoassay and proximal linkage immunoassay.

[0939] In the present invention, any of such principles of immunoassaymay be selected properly in accordance with the purpose. However, withconsideration given to the convenience of procedure and/or economicadvantage, and particularly clinical versatility, the principle ofsandwich method, one-pot method, or solid-phase one-antibody method,more preferably sandwich method or one-pot method is preferablyutilized. Particularly preferred is sandwich method using multi-wellmicrotiter plate having many wells such as 96-well microplate, orone-pot method using beads on which polypeptide is immobilized and alsousing a counterpart labeled with enzyme such as peroxidase or withbiotin.

[0940] The human monoclonal antibodies of the invention capable ofbinding to human AILIM are of human origin, and therefore these antibodyinduces no serious immunorejection due to antigenicity to human, i.e.,HAMA (Human anti-mouse antigenicity) in a host, which has been a serioustherapeutic problem (side effect) in antibody pharmaceuticalpreparations comprising non-human mammal-derived antibody such asmouse-derived antibody. The antibody of the invention is thus of greatvalue as an antibody pharmaceutical.

[0941] Thus, the human monoclonal antibody of the invention againstAILIM (particularly human AILIM) and pharmaceutical compositionscomprising the human monoclonal antibody do not induce hostimmunorejection caused by HAMA at all; and thus can be used aspharmaceutical preparations capable of controlling a variety ofbiological reactions (e.g., proliferation of cells expressing AILIM,cytokine production by AILIM-expressing cells, immune cytolysis or celldeath (apoptosis) of cells expressing AILIM, and activity of inducingantibody-dependent damage of cells expressing AILIM) associated with thetransduction of AILIM-mediated costimulatory signal (secondary signal)to cells expressing AILIM; and/or can be used to treat or preventvarious diseases associated with the transduction of the AILIM-mediatedsignal, controlling and inhibiting the onset and/or progress of thediseases.

[0942] Specifically, by providing pharmaceutical preparations containingthe human anti-AILIM monoclonal antibody of the invention or a portionthereof as an active ingredient, it is possible to inhibit or treat andprevent, for example, a variety of diseases (e.g., rheumatoid arthritis,multiple sclerosis, autoimmune thyroiditis, allergic contact dermatitis,lichen planus as a chronic inflammatory skin disease, systemic lupuserythematosus, insulin dependent diabetes mellitus and psoriasis, etc.)classified into autoimmune diseases or allergic diseases (particularly,autoimmune diseases and delayed allergies by cellular immunity);arthropathies (e.g., rheumatoid arthritis (RA), osteoarthritis (OA)),inflammation (e.g., hepatitis); graft versus host reaction (GVHreaction); graft versus host disease (graft versus host disease; GVHD);immunorejection associated with transplantation (allogenic graft orheterogenous graft) of tissues (tissues such as skin, cornea and bone)or organs (liver, heart, lung, kidney, pancreas, etc.); immune responseto foreign antigen or self antigen (for example, production of antibodyagainst the antigen, cell proliferation, cytokine production, etc.); anddiseases that are potentially caused by abnormality in gut immunity(specifically, inflammatory bowel disease (particularly, Crohn's diseaseand ulcerative colitis); and alimentary allergy, etc.

[0943] The pharmaceutical compositions in accordance with the presentinvention make it possible to treat or prevent some inflammations forwhich various steroidal drugs are used as anti-inflammatory drugs, forexample, inflammation associated with various arthritides (rheumatoidarthritis, osteoarthritis, etc.), pneumonia, hepatitis (including viralhepatitis), inflammation associated with infectious diseases,inflammatory bowel disease, enteritis, nephritis (glomerular nephritis,inflammation associated with kidney fibrosis, gastritis, vasculitis,pancreatitis, peritonitis, bronchitis, myocarditis, encephalitis,inflammation associated with ischemia-reperfusion injury (myocaridialischemia-reperfusion injury, etc.), inflammation associated withimmunorejection after transplantation of tissues or organs, scald,various skin inflammations (psoriasis, allergic contact dermatitis,lichen planus as a chronic inflammatory skin disease), inflammationassociated with multiple organ failure, inflammation after operation ofPTCA or PTCR, and inflammation associated with atherosclerosis,autoimmune thyroiditis, etc.

[0944] In addition, with respect to the above-mentioned inhibition andtreatment of immunorejection associated with transplantation of tissuesor organs as described above, the pharmaceutical compositions inaccordance with the present invention can be used in conjunction withknown immunosuppressant used to inhibit the immunorejection intransplantation therapy thereby increasing the effect of the known drugto inhibit the graft rejection.

[0945] Further, by the use of the method of identifying substancescapable of binding to AILIM or AILIM ligand, which is within the scopeof the present invention, it is possible to control the signaltransduction associated with the interaction between AILIM and AILIMligand through the binding to AILIM or AILIM ligand, and therebyachieving screening and selection of pharmaceutical agents (syntheticchemical compound and antibody) having potential activity to treat theabove-mentioned various diseases.

[0946] The present invention is illustrated in more detail below withreference to Examples, but is not to be construed as being limitedthereto.

EXAMPLE 1

[0947] Preparation of Immunogen

[0948] <1-1> Preparation of Recombinant Cell Expressing Human AILIM

[0949] Two types of recombinant cells (CHO cell and HPB-ALL cell)overexpressing human AILIM were prepared according to the method asdescribed in earlier applications (JP-A No. Hei 11-29599 andWO98/38216), as well as in a previous report (Int. Immunology, Vol.12,No.1, p.51-55, 2000) of one of the present inventors, Tezuka.Specifically, the method is as follows:

[0950] A cDNA (GenBank Accession Number: AB023 135 (cDNA); BAA82129(amino acid)) containing the full-length ORF encoding human AILIM wasinserted into a vector pEF-neo. Then the resulting recombinantexpression vector was introduced into Chinese hamster ovary cells (CHOcell) and cells of a human thymoma line, HPB-ALL, according to acommonly used method using electroporation (960 μF, 320V) with a GenePulser (BioRad). Respective cells were cultured in RPMI1640 mediumcontaining Geneticin (0.8 mg/ml; Gibco BRL) and 10% FCS to selectdrug-resistant transformed cells.

[0951] <1-2> Selection of Recombinant HPB-ALL Cells Overexpressing HumanAILIM

[0952] Culture of the drug-resistant HPB-ALL cells selected in <1-1>described above were centrifuged to give cell pellets. A mouseanti-human AILIM monoclonal antibody named “SA12” (mouse anti-humanJTT-1 antigen monoclonal antibody), which had been established andreported previously (JP-A 11-29599 (Example 12) and WO98/38216 (Example12)) by the present inventors, was added to the cell pellet(concentration: antibody solution (10 μg/ml) diluted with EDTA-BSA/PBSwas added at a ratio of 100 μl/10⁵ cells). The resulting mixtures wereincubated at 4° C. for 30 minutes. The cells were washed twice withabove-mentioned EDTA-BSA/PBS (200 ml), and then phycoerythrin-labeledstreptavidin (SA-PE; 100 μl of 500-fold diluted solution) was addedthereto. Resulting mixtures were incubated at 4° C. for 30 minutes.After incubation, cells were washed 3 times with EDTA-BSA/PBS, and cellsuspensions were prepared.

[0953] Expression levels of human AILIM of respective cells in the cellsuspensions were analyzed in a flow cytometer, FACSort(Beckton-Dichinson), to select recombinant HPB-ALL cells overexpressinghuman AILIM. Selected cells were cultured to confluence in RPMI1640medium containing 10% FCS and G418 (1 mg/ml).

[0954] <1-3> Selection of Recombinant CHO Cells Overexpressing HumanAILIM

[0955] Culture of the drug-resistant CHO cells selected in <1-1>described above were centrifuged to give cell pellets. Theabove-mentioned mouse anti-human AILIM monoclonal antibody SA12, whichhad been labeled with FITC, was added to each cell pellet (antibodysolution (100 μg/ml) diluted with EDTA-BSA/PBS). Resulting mixtures wereincubated at 4° C. for 30 minutes. Cells were washed withabove-mentioned EDTA-BSA/PBS, and then cell suspensions were prepared byadding EDTA-BSA/PBS (500 μl) to the cell pellets.

[0956] Expression levels of human AILIM of respective cells in the cellsuspensions were analyzed in a flow cytometer, FACSort(Beckton-Dichinson), to select recombinant HPB-ALL cells overexpressinghuman AILIM. Selected cells were cultured to confluence in RPM11640medium containing 10% FCS and G418 (1 mg/ml).

[0957] <1-4> Preparation of Immunogen from HPB-ALL Cells OverexpressingHuman AILIM

[0958] The HPB-ALL cells overexpressing human AILIM, which had beenobtained in <1-2> described above, were centrifuged. Recovered cellpellet was washed 4 times with phosphate buffer (PBS; Nikken Seibutsu)and then resuspended in a protease inhibitor-containing buffer(containing 25 mM HEPES (pH 7.4), 10 mM MgCl₂, 0.25 M Sucrose, andprotease inhibitor (10 U/ml Aprotinine, 2 μg/ml Pepstatin, 50 μg/mlLeupeptin, and 0.35 mg/ml PMSF)). Cell suspension was treated in aPotter-type homogenizer, and centrifuged at a low speed (at 1,500 rpm at4° C. for 10 minutes). Subsequently, resulting supernatant was subjectedto ultracentrifugation (under 100,000 g at 4° C. for 1 hour).Precipitated membrane fraction was recovered, and suspended in phosphatebuffer (concentration of the membrane fraction was adjusted so that 1 mlPBS contains membrane fraction derived from 1×10⁷ cells). The suspensionwas stored at −80° C. The suspension containing cell membrane fractionwas used as the antigen (immunogen) to prepare human antibody of thepresent invention, which will be described later.

[0959] <1-5> Preparation of Immunogen from CHO Cells OverexpressingHuman AILIM

[0960] The CHO cells overexpressing human AILIM, which had been obtainedin <1-3> described above, were dispersed with a scraper and werecentrifuged. Recovered cell pellet was washed 4 times with phosphatebuffer (PBS; Nikken Seibutsu) and then resuspended in a proteaseinhibitor-containing buffer (containing 25 mM HEPES (pH 7.4), 10 mMMgCl₂, 0.25 M Sucrose, and protease inhibitor (10U/ml Aprotinine, 2μg/ml Pepstatin, 50 μg/ml Leupeptin, and 0.35 mg/ml PMSF)). The cellsuspension was treated in a Potter-type homogenizer, and centrifuged ata low speed (at 1,500 rpm at 4° C. for 10 minutes). Subsequently,resulting supernatant was subjected to ultracentrifugation (under100,000 g at 4° C. for 1 hour). Precipitated membrane fraction wasrecovered, and suspended in phosphate buffer (concentration of themembrane fraction was adjusted so that 1 ml PBS contains membranefraction derived from 1×10⁷ cells). The suspension was stored at −80° C.The suspension containing cell membrane fraction was used as the antigen(immunogen) to prepare human antibody of the present invention, whichwill be described later.

EXAMPLE 2

[0961] Preparation of Hybridoma Producing Human Anti-human AILIMMonoclonal Antibody

[0962] In the present Example, preparation of monoclonal antibody wascarried out according to a typical method as described in “ExperimentalMedicine (supplement), Handbook for Cell Technology” (eds., T. Kuroki etal., Yodosha, pp.66-74, 1992) and “Experimental Manual for MonoclonalAntibody” (T. Ando et al., Kodansha, 1991).

[0963] Cell membrane fraction prepared from recombinant cellsoverexpressing human AILIM provided in Example 1 was used as theimmunogen of human AILIM.

[0964] Animals subjected to immunization were human antibody-producingtransgenic mice created by above-described method (Nature Genetics,Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997;Published Japanese Translation of International Application No. Hei4-504365; Published Japanese Translation of International ApplicationNo. Hei 7-509137; Nikkei Science, June, pp. 40-50, 1995; etc.).

[0965] Multi-well microplates were used for cell culture.

[0966] <2-1> Immunization and Preparation of Hybridoma

[0967] Either of the immunogens (100 μl/mouse/administration) preparedin <1-4> (derived from HPB-ALL) and in <1-5> (derived from CHO)described above was given to the above-mentioned humanantibody-producing transgenic mouse. The immunogen was injected togetherwit Freund's complete adjuvant (ICN/CAPPEL) in the footpad as primaryimmunization (day 0).

[0968] After primary immunization, either of the two immunogens wasadditionally injected to the footpad at 1-week interval as secondaryand/or tertiary immunization. In the same manner, injection was furthercarried out for final immunization two days before preparation oflymphocytes, which is described below.

[0969] Two days after final immunization, lymphocytes were prepared from(subinguinal and subgenual) lymph nodes and spleens of respectivetransgenic mice subjected to immunization. The lymphocytes and mousemyeloma cells P3/X63-AG8.653 (ATCC No. CRL-1580) were mixed at a ratioof 5:1, and polyethylene glycol 1500 (Boehringer Mannheim) was addedthereto as a fusion agent. Then, the mixture was diluted with 10 volumesof serum-free basal medium EX-CELL301 (JRH Bioscience). Subsequently,the mixed cells were washed with the basal medium and then suspended inHAT medium (1 L of basal medium contained 13.61 mg of hypoxanthine, 176μg of aminopterin, and 3.88 mg of thymidine). The cells were plated on96-well microplates and cultured for 10-14 days to complete cell fusion.The cell fusion treatment yielded many hybridomas.

[0970] <2-2> Screening of Human Monoclonal Antibody-producing Hybridoma

[0971] A number of hybridomas prepared in <2-1> described above werescreened with cell ELISA as described below to select hybridomasproducing human monoclonal antibody against human AILIM.

[0972] Respective recombinant HPB-ALL cells and recombinant CHO cellsoverexpressing human AILIM, which are described above, were plated ineach well of ELISA 96-well microplates (1×10⁵ cell/well). Incubation wascarried out at 37° C. for 2 days.

[0973] Subsequently, supernatant of each well was discarded, samples ofsupernatant of each hybridoma culture was added thereto (50 μl/well),and the mixture was incubated for 1 hour. After the reaction wascompleted, the mixed sample solution was discarded and each well waswashed 3 times with PBS containing 1% BSA (Sigma).

[0974] Subsequently, peroxidase-conjugated goat anti-humanimmunoglobulin (Fc) antibody (50 μl of the 2000-fold dilute per well;Ameircan Corex; 1% BSA/PBS) was added to each well in order to detectthe heavy chain of human immunoglobulin (human monoclonal antibody) inthe hybridoma supernatant. The mixture was incubated at room temperaturefor 1 hour.

[0975] On the other hand, peroxidase-conjugated goat anti-humanimmunoglobulin κ chain antibody (50 μl of the 2000-fold dilute per well)was added to each well in order to detect the light chain of humanimmunoglobulin (human monoclonal antibody) in the hybridoma supernatant.The mixture was incubated at room temperature for 15 minutes.

[0976] The anti-human IgFc antibody or anti-human Igκ antibody wasremoved from each well of the microplates, and then the plates werewashed 3 times with PBS containing 1% BSA. Tetramethylbenzidine(3,3′,5,5′,-tetramethylbenzidine (TMB), 100 μl/well, BIO-RAD) was addedto each well, and the resulting mixture was incubated at roomtemperature for 15 minutes.

[0977] Subsequently, 1N H₂SO₄ was added to each well (50 μl/well) toquench the reaction. The reaction was monitored for absorbance at awavelength of 450 mn by a microplate reader (Model 3550 MicroplateReader, BIO-RAD).

[0978] Control ELISA experiment was performed in the same manner asdescribed above by using the following items:

[0979] (1) Wild-type HPB-ALL cells, instead of human AILIM expressingrecombinant HPB-ALL cells;

[0980] (2) Wild-type CHO cells, instead of human AILIM expressingrecombinant CHO cells;

[0981] (3) Mouse monoclonal antibodies against human AILIM (SA12 orSG430; JP-A 11-29599 (Example 12) and WO98/38216 (Example 12)) insteadof hybridoma supernatant;

[0982] (4) Human monoclonal antibody against KLH (keyhole limpethemocyanin, PIERCE) instead of hybridoma supernatant.

[0983] The human anti-KLH monoclonal antibody was prepared according tothe same manner as described above in <2-1>, by immunizingabove-mentioned human antibody-producing transgenic mice with KLH(keyhole limpet hemocyanin, PIERCE).

[0984] Many hybridomas producing human monoclonal antibody capable ofbinding to human AILIM were selected by said screening.

[0985] <2-3> Primary Cloning of Hybridoma

[0986] Many types of hybridoma monoclones were established from thevarious hybridomas (parental cell lines), which had been selected in<2-2> described above, producing human monoclonal antibody against humanAILIM by the following assay procedure.

[0987] Respective hybridomas selected in <2-2> described above wereplated in 24-well microplates. Cell count of hybridoma in each well wasdetermined by pipetting. Subsequently, 10% fetal calf serum (FCS; TraceBioscience PTY), 1% penicillin/streptomycin (Sigma), 1% HT Supplement(Gibco BRL) and 2.5% T-STIM Culture Supplement (Collaborative BiomedicalProducts) were added to EX-CELL301 medium (JRH Bioscience) containing4.0 mM L-glutamine and lipid. The resulting modified medium was used todilute hybridomas to 1×10⁴ cells/ml and the cells were suspended in eachwell.

[0988] Cell suspension (300 μl or 600 μl) of each well was combined andmixed well with the modified medium (150 ml or 300 ml), and then a200-μl aliquot of the cell suspension was added to each well of multiple96-well microplates such that each well contained 4 cells of thehybridoma. The above-mentioned modified medium (50 ml or 100 ml) wasfreshly added to the remaining cell suspension, which was mixed well,and then the resulting cell suspension was added to each well of otherfreshly prepared multiple 96-well microplates such that each wellcontained 2 cells of the hybridoma.

[0989] Cultivation was continued for 1 to 2 weeks. After cultivation,single colonies derived from a single hybridoma were found in manywells.

[0990] With the cell ELISA as described above in <2-2>, it was verifiedthat human monoclonal antibody against human AILIM was produced in theculture supernatant in each well containing the colony.

[0991] <2-4> Secondary Cloning of Hybridoma

[0992] Subcloning (secondary cloning) of each clone from the varioushybridoma clones obtained in <2-3> described above was performedaccording to the same method as described above in <2-3>.

[0993] Cell density of each well in a 96-well microplate was adjusted to1 cell/well in the present experiment.

[0994] The screening yielded many hybridoma monoclones producing humanmonoclonal antibody against human AILIM. Part of the clones includedwere as below:

[0995] (Clone name)

[0996] AIF34 (JMab124), AIF182 (JMab-126), AIF348 (JMab-127),

[0997] AIF620 (JMab-128), AIF1052 (JMab-135), AIH5D3 (JMab-136),

[0998] AIH386 (JMab-137), AII289 (JMab-138), AII394 (JMab-139),

[0999] AII488 (JMab-140), AIJ40 (JMab-141),

[1000] The names shown above are used through the present application,namely in all Examples described below including the present Example,and in Figures and Tables containing the assay result obtained in thisExample.

EXAMPLE 3

[1001] Analysis of Properties of Monoclonal Antibody

[1002] <3-1> Analyses of the Heavy Chain and Light Chain

[1003] By using ELISA and flow cytometry described below, it wasverified that the monoclonal antibody against human AILIM produced byeach hybridoma clone, which had been cloned in <2-4> described above,was indeed a human monoclonal antibody.

[1004] The recombinant HPB-ALL cells overexpressing human AILIM, whichhad been prepared in Example 1, were plated in each v-shaped well ofmicroplate (3×10⁴ cell/well). Cells were cultured at 37° C. in RPMI1640medium containing 10% FCS.

[1005] After the culture was completed, the plate was centrifuged (at1,800 rpm for 2 minutes) to precipitate the cells, and then theresulting supernatant was discarded. Subsequently, supernatant sample(50 ml/well) from the culture of each hybridoma cloned in <2-4>described above, or mouse anti-human AILIM monoclonal antibody SA12 (2μg/50 μl) or alternatively human anti-KLH monoclonal antibody (50μl/well) as a control antibody was added to each well. The mixture wasreacted for 30 minutes in a refrigerator. After reaction, the samplesolution was discarded and each well was washed with phosphate buffer(0.5% BSA-PBS containing 5 mM EDTA).

[1006] Subsequently, any one of the secondary antibodies below was addedto each well (diluted 1000 times with the above phosphate buffer andadded at a quantity of 50 μl/well) in order to suspend the cells. Thesuspension was reacted for 30 minutes in a refrigerator. (Secondaryantibody)

[1007] Biotin-labeled anti-human IgG antibody (Zymed);

[1008] Biotin-labeled anti-human IgG antibody (Protos);

[1009] Biotin-labeled anti-human IgFc antibody (EY Laboratories); or

[1010] Biotin-labeled anti-human Igκ antibody (Vector).

[1011] After reaction, the secondary antibody was discarded and eachwell of the plate was washed with the above-mentioned phosphate buffer.Subsequently, phycoerythrin-labeled streptavidin (Streptavidin-PE;Pharmingen; diluted 500 time with the above-mentioned phosphate bufferand added at a quantity of 50 μl/well) was added to each well. Themixture was reacted for 30 minutes in a refrigerator. After reaction,each well was washed with the above-mentioned phosphate buffer. Then,the above-mentioned phosphate buffer was added to each well (200μl/well) in order to suspend the cells.

[1012] Analysis was performed to determine the reactivity of theanti-human AILIM monoclonal antibody in the culture supernatant of eachhybridoma clone to the HPB-ALL cells overexpressing human AILIM in eachwell.

[1013] Control assay was performed in the same manner as described aboveby using following items:

[1014] (1) Wild-type HPB-ALL cells, instead of human AILIM expressingrecombinant HPB-ALL cells;

[1015] (2) Human monoclonal antibody against KLH (keyhole limpethemocyanin, PIERCE) instead of hybridoma supernatant.

[1016] The human anti-KLH monoclonal antibody was prepared according tothe same manner as described above in <2-1>, by immunizing theabove-mentioned human antibody-producing transgenic mice with KLH(keyhole limpet hemocyanin, PIERCE).

[1017] Based on the result, all of the hybridoma clones described abovein <2-4> were verified to be human monoclonal antibodies consisting ofhuman-derived heavy chain and human-derived κ light chain.

[1018] An example of the result is illustrated in FIG. 1, which involvesassay result for hybridoma clones AIH5D3 (JMab-136), AII289 (JMab-138),and AII394 (JMab-139).

[1019] <3-2> Isotyping of Human Monoclonal Antibody

[1020] The isotype was determined for each of the human anti-human AILIMmonoclonal antibodies produced by the hybridomas that had been cloned in<2-4> and analyzed in <3-1> described above. Determination was carriedout using a Human Monoclonal Antibody Isotyping Kit (American Qualex)according to the experimental protocol attached to the kit.

[1021] All the human anti-human AILIM monoclonal antibodies weredetermined to be IgG2/κ.

EXAMPLE 4

[1022] Preparation of Human Monoclonal Antibody Against Human AILIM(Human Anti-human AILIM Monoclonal Antibody) on Large Scale and itsPurification

[1023] <4-1> Method 1

[1024] Cells of each hybridoma clone producing human anti-human AILIMmonoclonal antibody, which had been prepared in <2-4> described above,were added to a tissue culture flask (50 ml, FALCON), and cultured inASF104 medium (Ajinomoto) containing 10% Ultra Low Bovine IgG FBS(GIBCO-BRL) to be confluence under an atmosphere of 5% CO₂ at 37° C.

[1025] Subsequently, the whole culture liquid was transferred into a newtissue culture flask (750 ml, FALCON), and the cells were cultured inASF104 medium (Ajinomoto) containing 10% Ultra Low Bovine IgG FBS(GIBCO-BRL) to be confluent under an atmosphere of 5% CO₂ at 37° C.

[1026] 10 to 20 days after the culture, the culture supernatant of eachhybridoma was recovered and transferred into a 50-ml polypropyleneconical tube (FALCON). The tube was centrifuged under 500×g for 5minutes.

[1027] Subsequently, resulting centrifugal supernatant was filteredthrough a Sterilization Filter Unit (NALGEN), and the filtrate wasrecovered.

[1028] The filtrate was loaded onto a HiTrap Protein G column (HiTrapaffinity column Protein G; Amersham Pharmacia) pre-equilibrated withphosphate buffer (30 ml) at a flow rate of 3 ml/min.

[1029] Subsequently, the column was washed with phosphate buffer (20ml), and then the antibody of interest was eluted by loading 100 mMcitrate buffer (pH 2.0) onto the column at a low rate of about 1 ml/min.Subsequently, the eluted solution was neutralized with a solution (pH9.0) of 750 mM Tris-HCl, and then filtered through a filter (Millipore)to remove white precipitate. The resulting filtrate was dialyzed againstphosphate buffer (overnight) and filtered through a filter (Millipore).Thus purified anti-AILIM human monoclonal antibody was obtained fromeach hybridoma line.

[1030] Protein concentration was determined from the absorbance at A₂₈₀measured by using a photospectrometer (1A₂₈₀=1.41 mg/ml).

[1031] <4-2> Method 2

[1032] Cells of each hybridoma clone, which had been prepared in <2-4>described above, were conditioned in ASF104 medium (Ajinomoto)containing 10% Ultra Low Bovine IgG FBS (GIBCO-BRL) (1-2×10⁶ Cells/mleach), and were plated and cultured in Integra Cell Line 1000 (INTEGRACL1000, INTEGRA BIOSCIENCE). 7 to 10 days after cultivation, when thecell density reached 1×10⁸ cells/ml, the supernatant of each hybridomaculture was recovered.

[1033] Each culture supernatant was loaded onto a HiTrap Protein Gcolumn (HiTrap affinity column Protein G; Amersham Pharmacia)pre-equilibrated with phosphate buffer (30 ml) at a flow rate of 3ml/min.

[1034] Subsequently, the column was washed with phosphate buffer (20ml), and then 100 mM citrate buffer (pH 2.0) was loaded onto the columnat a flow rate of about 1 ml/min to elute the antibody. Then, a solution(pH 9.0) of 750 mM Tris-HCl was added to neutralize the eluted solution,and the resulting solution was filtered through a filter (Millipore) toremove white precipitate. The resulting filtrate was dialyzed againstphosphate buffer (overnight) and then filtered through a filter(Millipore). Thus purified anti-AILIM human monoclonal antibody wasobtained from each hybridoma line.

EXAMPLE 5

[1035] Reactivity of Human Anti-human AILIM Monoclonal Antibody to HumanAILIM, and Cross-reactivity of that to Mouse AILIM and Rat AILIM

[1036] Purified various human anti-human AILIM monoclonal antibodiesabove were analyzed for their reactivity to human AILIM as well ascross-reactivity to mouse AILIM and rat AILIM by utilizing cell ELISAmethod.

[1037] <5-1> Establishment of ELISA System to Determine IgG AntibodyConcentration and Preparation of Calibration Curves

[1038] Because all the above-mentioned purified human anti-human AILIMmonoclonal antibodies were IgG (IgG2) antibody, ELISA system wasestablished to determine the concentration of IgG antibody.

[1039] Goat anti-human IgG (Fc) antibody (1.2 μg/ml in PBS; 100 μl/well;Organon Teknika) was added to each well of a 96-well ELISA microplate(Nunc). The plate was incubated at room temperature for 2 hours toadsorb the anti-IgG (Fc) antibody on the microplate. Subsequently, thesupernatant was discarded, and the plate was washed 3 times withphosphate buffer (PBS) containing 0.05% Tween20. A blocking reagent (PBScontaining 0.5% bovine serum albumin (BSA) and 0.1% Tween20) was addedto each well (200 μl/well) and the plate was incubated at roomtemperature for 2 hours to block the anti-IgG (Fc) antibody-free siteson the plate. Then, the blocking reagent was discarded, and each wellwas washed twice with PBS.

[1040] Human-derived IgG2 antibody (50 μl/well; The Binding Site), whichwas used as a standard antibody, was added at various concentrations (0to 100 ng/ml) to respective wells of the plate and the plate wasincubated at room temperature for 2 hours. Surplus solutions of standardantibody were removed and each well was washed 3 times with phosphatebuffer containing 0.05% Tween20.

[1041] Subsequently, peroxidase-conjugated goat anti-human IgG/κantibody was added to each well (4,000 times diluted, 100 μl/well,Protos), and the plate was incubated at room temperature for 1 hour.

[1042] The supernatant was discarded and the microplate was washed 3times with phosphate buffer containing 0.05% Tween20. A buffercontaining substrate (composition: ortho-phenylenediamine(O-phenylenediamine, OPD; 20 mg)/citrate-phosphate buffer (pH 5.0, 50ml)/aqueous solution of 30% hydrogen peroxide (15 μl)) was added to eachwell (100 μl/well) and the plate was incubated at room temperature forabout 7 minutes.

[1043] Subsequently, 2 M sulfuric acid was added to each well (50μl/well) to stop the reaction. Calibration curves were made (FIG. 2)based on the values of absorbance measured at a wavelength of 490 nm byusing a microplate reader.

[1044] Control assays were performed with culture medium alone or BSAsolution alone as a test substance in the same manner as describedabove.

[1045] <5-2> Analyses for the Reactivity of Various Purified HumanAnti-human AILIM Monoclonal Antibodies to Human AILIM as Well as forCross-reactivity of that to Mouse AILIM and Rat AILIM

[1046] <5-2-1> Preparation of Reagents

[1047] Reagents to be used in this cell ELISA were prepared as follows:

[1048] <5-2-1-1> Preparation of Recombinant CHO Cell OverexpressingMouse AILIM

[1049] Recombinant CHO cells overexpressing mouse AILIM were preparedand obtained in the same manner as described above in <1-1> and <1-3>.

[1050] cDNA (GenBank Accession Number: AB023132 (cDNA); BAA82126 (aminoacid)) containing the full-length ORF of mouse AILIM was inserted into avector pEF-neo, and then the resulting recombinant expression vector wasintroduced into Chinese hamster ovary cells (CHO cell) by a commonlyused method for electroporation (960 μF, 320 V) using a Gene Pulser(BioRad). The cells were cultured in RPMI1640 medium containingGeneticin (0.8 mg/ml; Gibco BRL) and 10% FCS to select drug-resistanttransformed cells, thereby obtaining mouse AILIM-overexpressingrecombinant CHO cells.

[1051] <5-2-1-2> Preparation of Recombinant CHO Cell Overexpressing RatAILIM

[1052] Recombinant CHO cells overexpressing rat AILIM were prepared andobtained in the same manner as described above in <1-1> and <1-3>.

[1053] A cDNA (GenBank Accession Number: AB023134 (cDNA); BAA82128(amino acid)) containing the full-length ORF of rat AILIM was insertedinto a vector pEF-neo, and then the resulting recombinant expressionvector was introduced into Chinese hamster ovary cells (CHO cell) by acommonly used method for electroporation (960 μF, 320 V) using a GenePulser (BioRad). The cells were cultured in RPMI1640 medium containingGeneticin (0.8 mg/ml; Gibco BRL) and 10% FCS to select drug-resistanttransformed cells, thereby obtaining rat AILIM-overexpressingrecombinant CHO cells.

[1054] <5-2-1-3> Preparation of Monoclonal Antibody Against Mouse AILIM

[1055] The recombinant CHO cells overexpressing mouse AILIM, which hadbeen prepared in <5-2-1-1> described above were homogenized, andsubjected to ultracentrifugation (100,000×g). Resulting pelletcontaining cell membrane fraction was recovered and then suspended inPBS. Resulting cell membrane fraction was injected together withFreund's complete adjuvant to Wistar rats in the footpad for primaryimmunization (day 0). The antigen of cell membrane fraction was furthergiven to the rats into the footpad on the 7^(th) day, 14^(th) day and28^(th) day after the primary immunization. The lymph node cells werecollected from them 2 days after the final immunization.

[1056] The lymph node cells and mouse myeloma cell PAI (JCR No.B0113;Res. Disclosure, Vol.217, p.155, 1982) were combined at a ratio of 5:1.The cells were fused to each other by using polyethylene glycol 4000(Boehringer Mannheim) as a fusion agent to prepare monoclonalantibody-producing hybridomas. Selection of hybridomas was achieved byculturing them in ASF104 medium (Ajinomoto) containing HAT, 10% fetalcalf serum and aminopterin.

[1057] Reactivity of rat monoclonal antibody in the culture supernatantof each hybridoma to mouse AILIM was determined by reacting the culturesupernatant with the above-mentioned CHO cells expressing mouse AILIMand then measuring the fluorescence intensity of cells stained withFITC-labeled anti-rat IgG (Cappel) in a EPICS-ELITE flow cytometer. Thescreening yielded multiple hybridomas producing monoclonal antibodyhaving reactivity against mouse AILIM.

[1058] Among them, a hybridoma line was named “B10.5.” Cells of thishybridoma were intraperitoneally injected (10⁶ to 10⁷ cells/0.5ml/mouse) to ICR nu/nu mice (female, 7 to 8-weeks old). 10 to 20 daysafter the injection, the ascites was collected from each mouse bylaparotomy under anesthesia according to a commonly used method. The ratanti-mouse AILIM monoclonal antibody B10.5 (IgG1) was prepared from theascites on a large scale.

[1059] <5-2-2> The Reactivity of the Antibody to Human, Mouse and RatAILIM

[1060] Concentrations of human anti-human AILIM monoclonal antibody andcontrol antibody to be used in the ELISA described below were determinedbased on the ELISA and calibration curves in <5-1> described above.

[1061] Each cells (7×10³ cells/well) of the recombinant CHO celloverexpressing human AILIM prepared in Example 1, the recombinant CHOcell overexpressing mouse AILIM prepared in <5-2-1-1> described above,and the recombinant CHO cell overexpressing rat AILIM prepared in<5-2-1-2> described above were plated in wells of 96-well ELISAmicroplates and cultured to be confluent at 37° C.

[1062] Subsequently, the supernatant was discarded, and then any one ofthe purified various human anti-human AILIM monoclonal antibodies orcontrol antibody prepared above were added to each well (antibodyconcentrations: antibody of 200 μg/ml was diluted, with PBS containing1% BSA, 3 times, 3² times, 3³ times, 3⁴ times, 3⁵ times, 3⁶ times, 3⁷times, 3⁸ times, 3⁹ times, 3¹⁰ times, 3¹¹ times, and 3¹² times) in aquantity of 50 μl/well, and the plates were reacted at room temperaturefor 2 hours. The solutions of the monoclonal antibodies were discarded,and each well was washed 3 times with phosphate buffer containing 1% BSA(Sigma).

[1063] Subsequently, horseradish peroxidase-conjugated anti-human IgG(Fc) antibody was added to each well (diluted 1,000 times, 50 μl/well;American Qualex), and the plates were incubated at room temperature for1 hour.

[1064] Surplus solution of the labeled antibody was discarded and themicroplates were washed 3 times with phosphate buffer containing 1% BSA.Buffer containing substrate (composition: ortho-phenylenediamine(O-phenylenediamine, OPD; 20 mg)/citrate-phosphate buffer (pH 5.0, 50ml)/aqueous solution of 30% hydrogen peroxide (15 μl)) was added to eachwell (100 μl/well) and the plates were incubated at room temperature forabout 7 minutes.

[1065] Subsequently, 2 M sulfuric acid was added to each well (50μl/well) to stop the reaction. Absorbance was measured at a wavelengthof 490 nm by using a microplate reader (Bio-Rad).

[1066] Control ELISA assay was performed with the following controlantibodies to evaluate the above-mentioned antibodies in the same manneras described above:

[1067] (1) Mouse monoclonal antibody SA12 or SG430 against human AILIM(JP-A 11-29599 (Example 12) and WO98/38216 (Example 12));

[1068] (2) Rat monoclonal antibody B10.5 against mouse AILIM (<5-2-1-3>described above);

[1069] (3) Mouse monoclonal antibody JTT2 against rat AILIM (monoclonalantibody produced by a hybridoma, which has been depositedinternationally on Oct. 11, 1996, under the international accessionnumber FERM BP-5708 in The National Institute of Bioscience andHuman-Technology, The Agency of Industrial Science and Technology, TheMinistry of International Trade and Industry)), which is aninternational depositary authority under the Budapest Treaty; JP-A11-29599 (Examples 1 and 2) and WO98/38216 (Examples 1 and 2)).

[1070] (4) The above-prepared human monoclonal antibody against KLH(keyhole limpet hemocyanin, PIERCE) instead of hybridoma supernatant.

[1071] Control ELISA experiment was performed in the same manner asdescribed above using wild-type CHO cell shown below, instead ofAILIM-expressing recombinant CHO cell.

[1072] The result is shown in FIGS. 3 to 14.

[1073] Based on the result obtained, 50%-effective concentration(ED50:ng/ml) was calculated as an index for the reactivity of each humananti-human AILIM monoclonal antibody to human AILIM (recombinant CHOcell overexpressing human AILIM), mouse AILIM (recombinant CHO cellsoverexpressing mouse AILIM), or rat AILIM (recombinant CHO celloverexpressing rat AILIM). The results obtained by the calculation areshown below. (A) ED50 for CHO over-expressing human AILIM AIF 34(JMab-124):  5.3 ng/ml AIF182 (JMab-126):  3.6 ng/ml AIF348 (JMab-127): 9.1 ng/ml AIF620 (JMab-128): 10.1 ng/ml AIF1052 (JMab-135):  2.0 ng/mlAIH5D3 (JMab-136):  7.5 ng/ml AIH386 (JMab-137):  9.6 ng/ml AII289(JMab-138): 10.5 ng/ml AII394 (JMab-139): 10.6 ng/ml AII488 (JMab-140):11.0 ng/ml AIJ 40 (JMab-141):  3.7 ng/ml SA 12:  1.8 ng/ml SG430:  1.2ng/ml

[1074] (B) ED50 for CHO overexpressing mouse AILIM AIF 34 (JMab-124): 42 ng/ml AIF348 (JMab-127):  81 ng/ml AIF620 (JMab-128): 100 ng/mlAII289 (JMab-138):  53 ng/ml AII394 (JMab-139):  60 ng/ml AII488(JMab-140):  70 ng/ml

[1075] (C) ED50 for CHO overexpressing rat AILIM AIF 34 (JMab-124): 45ng/ml AIF348 (JMab-127): 62 ng/ml AIF620 (JMab-128): 97 ng/ml AII289(JMab-138): 57 ng/ml AII394 (JMab-139): 90 ng/ml AII488 (JMab-140): 90ng/ml

[1076] The result showed that the human anti-human AILIM monoclonalantibodies of the present invention exhibited significantly highspecificities to human AILIM.

[1077] Further, it has been revealed that 6 types of human anti-humanAILIM monoclonal antibodies (shown above in (B) and (C)) are reactive toboth mouse AILIM and rat AILIM (binding capability, cross-reactivity).

EXAMPLE 6

[1078] Determination of Affinity and Neutralizing Activity of HumanAnti-human AILIM Monoclonal Antibody Against the Antigen (Human AILIM)

[1079] Association rate constant (ka), dissociation rate constant (kd)and dissociation constant (Kd) with respect to the reaction between eachof the purified various human anti-human AILIM monoclonal antibodiesprepared above and human AILIM were determined using a commerciallyavailable kit Biacore X (Amersham Pharmacia).

[1080] <6-1> Preparation of Antigen to be Immobilized on Sensor Chip

[1081] Antigen to be immobilized on sensor chip in the kit was preparedas a recombinant chimeric antigen (hereinafter referred to as “humanAILIM-IgFc”) consisting of the extracellular region of human AILIM andthe constant region (Fc) of human IgG1.

[1082] Human AILIM-IgFc was prepared by further purifying the antigenobtained according to the method as described in earlier applications(JP-A 11-29599 (Example 16 (2)) and WO98/38216 (Example 16 (2)) by oneof the present inventors, Tezuka.

[1083] The culture supernatant of recombinant cells producing the humanAILIM-IgFc was loaded onto a HiTrap Protein G column (HiTrap affinitycolumn Protein G; Amersham-Pharmacia) pre-equilibrated with phosphatebuffer (30 ml) at a flow rate of 3 ml/min to adsorb the human AILIM-IgFcin the culture supernatant on the column.

[1084] Subsequently, the column was washed with phosphate buffer (20ml), and then 100 mM citrate buffer (pH 2.0) was loaded onto the columnat a flow rate of about 1 ml/min to elute the human AILIM-IgFc.Subsequently, the eluted solution was neutralized with a solution (pH9.0) of 750 mM Tris-HCl, and then dialyzed against phosphate buffer(overnight). Then, the solution dialyzed was filtered through a filter(Millipore). Thus purified anti-human AILIM-IgFc was obtained.

[1085] Protein concentration was determined from the absorbance at A₂₈₀measured by using a photospectrometer(1A₂₈₀=1 mg/ml). The concentrationof human AILIM-IgFc was determined to be 0.28 mg/ml.

[1086] Purified chimeric protein consisting of the extracellular regionof rat AILIM and the constant region (Fc) of human IgG1 (rat AILIM-IgFc;JP-A 11-29599 (Example 16 (2)) and WO98/38216 (Example 16 (2)) was alsoprepared according to the same manner as described above. Theconcentration of the rat AILIM-IgFc obtained was determined to be 0.45mg/ml.

[1087] <6-2> Determination of affinity and neutralizing activity

[1088] Experimental procedures except for immobilization of antigen(human AILIM-IgFc) on the sensor chip, which is described below, werebased on the instruction manual and experimental protocol attached tothe commercially available assay kit Biacore X (Amersham-Pharmacia).

[1089] HBS buffer (containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA and0.005% detergent P20, (pH 7.0)) was allowed to flow through a FlowCell-1 attached to the kit at a flow rate of 5 μl/min. Subsequently, asolution (15 μl) containing 0.005 M NHS (N-hydroxysuccinimide) and 0.2 MEDC (N-ethyl-N′-(dimethylaminopropyl) carbodiimide) was added toactivate carboxyl groups of CM coated on the surface of the sensor chip.

[1090] Subsequently, 23 μl of human AILIM-IgFc solution (10 μg/ml;dissolved in 10 mM sodium acetate buffer (pH 5.0)) was added to the toimmobilize the human AILIM-IgFc on the sensor chip. Subsequently,unreacted activated carboxyl groups were blocked by adding 35 ml of 1 Methanol amine hydrochloride. The amounts of human AILIM-IgFc immobilizedby the immobilization treatment performed twice was 2,444RU (resonanceunit) and 2,213RU, respectively. The unit, RU, corresponds to the massper unit area; 1RU=1 pg/mm².

[1091] Flow Cell-2, which is a reference flow cell, was subjected to thecapping treatment in the absence of human AILIM-IgFc in the same manneras described above.

[1092] Phosphate buffer was allowed to flow through the flow cell(sensor chip) at a flow rate of 20 μl/min, and each of purified humananti-human AILIM monoclonal antibodies, which had been prepared in theExample above, was added thereto (10 to 50 μg/ml, 60 μl).

[1093] Standard condition for the measurement was: association phase for3 minutes and dissociation phase for 10 minutes. Respective amounts ofantibody bound to and released from the antigen were monitored over timeto obtain a sensorgram. Dissociation of antibody from the antigen wasachieved by running PBS through the sensor chip at a flow rate of 20μl/min.

[1094] Based on the resulting sensorgram data, association rate constant(ka), dissociation rate constant (kd) and dissociation constant (Kd;Kd=kd/ka) were computed by using the analytical software (BIAevaluation3.0) attached to the kit.

[1095] The affinity and the neutralizing activity of mouse monoclonalantibodies SA12 and SG430 to the human AILIM prepared in the Exampledescribed above were also analyzed in the same manner as describedabove. Respective values obtained are shown below. <clone name> <ka(1/M.Sec)> <kd [1/Sec]> <Kd (M)> AIF 34 (JMab-124) 1.6 × 10⁴ 1.0 × 10⁻⁴6.3 × 10⁻⁹ AIF182 (JMab-126) 3.2 × 10⁴ 2.8 × 10⁻⁵ 8.8 × 10⁻¹⁰ AIF348(JMab-127) 1.9 × 10⁴ 6.4 × 10⁻⁵ 3.4 × 10⁻⁹ AIF620 (JMab-128) 1.1 × 10⁴1.1 × 10⁻⁴ 1.0 × 10⁻⁸ AIF1052 (JMab-135) 1.6 × 10⁴ 6.3 × 10⁻⁵ 3.9 × 10⁻⁹AIH5D3 (JMab-136) 2.8 × 10⁴ 4.9 × 10⁻⁶ 1.8 × 10⁻¹⁰ AIH386 (JMab-137) 1.2× 10⁵ 3.1 × 10⁻⁴ 2.6 × 10⁻⁹ AII289 (JMab-138) 3.7 × 10⁴ 4.2 × 10⁻⁵ 1.1 ×10⁻⁹ AII394 (JMab-139) 3.1 × 10⁴ 2.4 × 10⁻⁵ 7.7 × 10⁻¹⁰ AII488(JMab-140) 2.3 × 10⁴ 3.5 × 10⁻⁵ 1.5 × 10⁻⁹ AIJ 40 (JMab-141) 1.9 × 10⁴1.9 × 10⁻⁵ 1.0 × 10⁻⁹ SA 12 7.8 × 103 7.9 × 10⁻⁵ 1.0 × 10⁻⁸ SG430 2.2 ×104 1.5 × 10⁻⁴ 6.8 × 10⁻⁹

[1096] The result shows that all of the human anti-human AILIMmonoclonal antibodies and anti-human AILIM mouse monoclonal antibodiesexhibit markedly high binding affinity and neutralizing activity tohuman AILIM.

EXAMPLE 7

[1097] Activity of Human Anti-human AILIM Monoclonal Antibody toTransduce Costimulatory Signal in Human T Cell

[1098] It was analyzed whether or not the human anti-human AILIMmonoclonal antibodies in accordance with the present invention had thecapability of controlling (enhancing and/or inhibiting) human T cellresponses (production of cytokines such as IFN-γ and IL-4, cellproliferation, etc.), in other words, whether or not the antibodiesexhibited regulatory activity on cellular transduction of AILIM-mediatedcostimulatory signal. Analysis was performed based on the amount ofcytokines (IFN-γ and IL-4) produced in human T cells as well as thedegree of human T cell proliferation as an index.

[1099] <7-1> Dilution of Antibody

[1100] Anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) wasdiluted with phosphate buffer (PBS) to final concentration of 8 μg/ml.

[1101] Each of the various human anti-human AILIM monoclonal antibodiesprepared above was diluted with PBS to a final concentration of 40μg/ml. The antibody solutions were further diluted with PBS to preparevarious concentrations of antibodies (40 μg/ml-0.0049 μg/ml).

[1102] <7-2> Coating of Microplate with Antibody

[1103] Each well of 96-well microplates was coated with (1) anti-humanCD3 monoclonal antibody OKT3 (8 μg/ml; 25 μl to each well) and any oneof the various human anti-human AILIM monoclonal antibodies (40μg/ml-0.0049 μg/ml; 25 μl to each well), or (2) anti-human CD3monoclonal antibody OKT3 (8 μg/ml; 25 μl to each well) alone. The plateswere incubated at 37° C. for 2 hours. Subsequently, the antibodysolutions were discarded, and each well was washed 3 times with PBS.After the wash, RPMI1640 medium containing 10% FCS was added to eachwell (100 μl/well), and the plates were incubated at 37° C. for 1 hour.Thus, respective wells of the plates were coated with the antibodiesmentioned above in (1) or (2).

[1104] Control experiments were carried out in the same manner by usingplates coated with the following respective monoclonal antibodies ascontrol antibodies instead of the human anti-human AILIM monoclonalantibodies.

[1105] (1) Mouse monoclonal antibody SA12 or SG430 against human AILIM(JP-A 11-29599 (Example 12) and WO98/38216 (Example 12));

[1106] (2) Mouse anti-human CETP monoclonal antibody JHC1 (also referredto as JMab109; JP-A 9-20800); and

[1107] (3) Human anti-KLH monoclonal antibody (also referred to asJMab23; the above-mentioned Example).

[1108] The microplates coated with the antibodies were used in thefollowing assays.

[1109] <7-3> Preparation of Human T Cell Suspension

[1110] Peripheral blood was collected from each normal healthy persons(5 persons; donor A, B, C, D and E). Fraction containing mononuclearcells was prepared by density-gradient centrifugation using LymphoPrep(Nycomed). Human T cells were separated from the human mononuclear cellfraction according to the manual for experimental procedure by using aPan-T cell Isolation Kit (Miltenyi) and Magnetic Sorter. T cell countwas determined using a hemacytometer. Human T cells were suspended inRPMI1640 medium containing 10% FCS to prepare human T cell suspension(1×10⁶ cell/ml).

[1111] <7-4> Cell Culture

[1112] (1) Culture Using Microplate Coated with Anti-human CD3 Antibodyand Anti-human AILIM Antibody

[1113] Human T cell suspension (donor A, B, C, D or E; 100 μl/well;1×10⁵ cells/well) was added to each well of the microplate coated withthe antibody mentioned above and the plate was incubated at 37° C. for 3days in a CO₂ incubator.

[1114] After cultivation, aliquots of the resulting culture supernatants(50 μl) were stored at −20° C. and then used in the assay describedlater (assay for IFNγ). After sampling aliquots of the culturesupernatants, respective microplates were used for the following assay:

[1115] (2) Culture Using Microplate Coated with Anti-human CD3 AntibodyAlone

[1116] Human T cell suspension (donor D; 100 μl/well; 1×10⁵ cells/well)was added to each well of the microplates coated with theabove-mentioned antibody, and then any one of the various humananti-human AILIM monoclonal antibodies was added thereto (25 μl of theantibody of 40 μg/ml-0.0049 μg/ml). The plates were incubated at 37° C.for 3 days in a CO₂ incubator.

[1117] <7-5> Determination of Proliferation Activity of T Cell

[1118] Methyl [³H]thymidine (0.5 μCi/well; Amersham-Pharmacia) was addedto each well of the plates after incubation, and the plates wereincubated at 37° C. for 6 hours in a CO₂ incubator. After incubation,cells were trapped on GF/C filters (Packard) using Cell Harvester.Subsequently, the filters were dried at 40° C. for 3 hours or longer,and then Microscinti 0 (20 μl/well; Packard) was added thereto.Radioactivity of ³H incorporated of the cells trapped on the filters wasmeasured by a β-counter (TOP COUNT) to analyze the degree of T cellproliferation after cultivation.

[1119] Results are shown in FIGS. 15 to 39.

[1120] Result of this assay showed that human T cells were significantlyproliferated depending on the concentration of the cells whenmicroplates were coated with anti-human AILIM monoclonal antibody (humanmonoclonal antibody or mouse monoclonal antibody) together withanti-human CD3 antibody. Further, there were some differences in thedegree of cell proliferation among the donors.

[1121] On the other hand, human T cells did not grow significantly whenplates had been coated with anti-human CD3 antibody alone and anti-humanAILIM monoclonal antibody (human monoclonal antibody or mouse monoclonalantibody) in solution (liquid phase) was used during culturing thecells.

[1122] <7-6> Quantification of IFNγ in Culture Supernatant of T Cell

[1123] For respective cultures of T cells (donors B and C) described in(1) of <7-4>, the amounts of IFNγ in the culture supernatants weredetermined by a commercially available human IFNγ ELISA KIT(Amersham-Pharmacia; Endogen).

[1124] Results are shown in FIGS. 40 to 47.

[1125] Result of this assay showed that the production of IFNγ increasedsignificantly depending on the concentration of anti-human AILIMmonoclonal antibody (human monoclonal antibody or mouse monoclonalantibody).

EXAMPLE 8

[1126] Regulatory Activity of Human Anti-human AILIM Monoclonal Antibodyon Mixed Lymphocyte Reaction (MLR)

[1127] It was tested whether or not the human anti-human AILIMmonoclonal antibodies of the present invention were capable ofcontrolling (enhancing and/or inhibiting) T cell responses (productionof cytokines such as IFN-γ and IL-4, cell proliferation, etc.), in otherwords, capable of regulating the transduction of AILIM-mediatedcostimulatory signal into cells, by analyzing the activity (namely, DNAsynthesis in cells) of controlling T cell proliferation associated withallogenic mixed lymphocyte reaction (allogenic MLR) as an index.

[1128] <8-1> Preparation of Human PBMC and T Cell

[1129] Peripheral blood (200 ml) collected from each normal healthypersons (7 persons; donor A, B, C, D, E, F and G) was dispensed on thelayers of Lymphoprep (15 ml; Nycomed) in microtubes (50 ml; Falcon).After centrifugation (at 1600 rpm for 10 minutes), intermediate layerswere recovered. Recovered cells were diluted 2 times or further withphosphate buffer, and then centrifuged (at 1,800 rpm for 10 minutes).Thus, PBMC (peripheral blood mononuclear cell; 2×10⁸-5×10⁸ cell) wasprepared. Cell count was determined by using a hemacytometer. An aliquotof the cells to be used in MLR assay (1.08×10⁸ cell/9 microplates) weretaken and kept on ice. Remaining cells were used for the separation of Tcells described below.

[1130] PanT Isolation kit (Miltenyi Biotech) was used for the separationof T cells from PBMC. According to the manual attached to the kit,remaining PBMCs were added to the solution attached to the kit, and thesolution was incubated. Subsequently, cells were washed with PBScontaining 5 mM EDTA and 0.5% BSA and then re-suspended in PBS.Subsequently, the cell suspension was added to a Positive SelectionColumn VS+ (Miltenyi Biotech) swollen with PBS, and unadsorbed fractionwas recovered. Further, PBS was loaded onto the column, and the washsolution was recovered. The same treatment was repeated once. Recoveredsolutions were combined together to give a T cell fraction. Aftercentrifugation of the T cell fraction, cells were re-suspended in PBS.Cell count of the resulting T cells was determined by using ahemacytometer. The cells were used in the following assay.

[1131] <8-2> Mixed Lymphocyte Reaction (MLR)

[1132] As described above, two signaling pathways one between CD28 andCD80(B7-1)/CD86(B7-2) and the other between CTLA4 andCD80(B7-1)/CD86(B7-2), for which comparatively detailed analysis havebeen previously made, are known as costimulatory signaling pathwaysrequired for the activation of lymphocytes such as T cell, etc.

[1133] Namely, the proliferation of T cell in response to mixedlymphocyte reaction (MLR) can be induced by the signal transductionthrough each of the two known pathways.

[1134] Thus, by using the substances indicated below, test of thisinvention was conducted to analyze (1) the inhibition of MLR by blockingthe CTLA4-mediated signaling pathway; (2) the inhibition of MLR byblocking the CD80 (B7-1)/CD86(B7-2)-mediated signaling pathway; (3) theinhibition of MLR by blocking both CTLA4-mediated pathway and CD80(B7-1)/CD86 (B7-2)-mediated signaling pathway; (4) the inhibition of MLRby blocking the tertiary signaling pathway associated with AILIM; and(5) the inhibition of MLR by blocking both CTLA4-mediated pathway andAILIM-mediated pathway.

[1135] Following test substances were used.

[1136] (1) Human anti-human AILIM monoclonal antibody (prepared in theExample described above);

[1137] (2) Mouse anti-human AILIM monoclonal antibody SA12 (same as inthe above Example);

[1138] (3) Human anti-KLH monoclonal antibody (negative control; same asin the above Example);

[1139] (4) Mouse IgG antibody (anti-human CD34; negative control;Immunotech);

[1140] (5) A mixture of anti-human CD80 monoclonal antibody (Pharmingen)and anti-human CD86 monoclonal antibody (Pharmingen); and

[1141] (6) Human CTLA4-IgFc chimera molecule (Ancell).

[1142] Mixed lymphocyte reaction (MLR) was conducted on the following 6combinations using PBMCs and T cells prepared from the donors describedabove in <8-1>. (i) T cell (donor A)/PBMC (donor D) (ii) T cell (donorD)/PBMC (donor B) (iii) T cell (donor C)/PBMC (donor A) (iv) T cell(donor E)/PBMC (donor G) (v) T cell (donor F)/PBMC (donor E) (vi) T cell(donor G)/PBMC (donor F)

[1143] The concentrations of PBMCs and T cells to be used in the testwere adjusted as described below.

[1144] PBMCs were suspended in PBS, and then transferred into culturedishes (60 mm). The cells were subjected to X-ray irradiation (50 Gy)with an irradiator (Hitachi MEDICO). Cells were recovered, centrifugedand then added to PRMI1640 medium containing 10% FCS. Cell count wasadjusted to 2×10⁵ cells/50 μl.

[1145] Resulting T cells from each donor were also added to PRMI1640medium containing 10% FCS and the cell count was adjusted to 1×10⁵cells/50 μl.

[1146] <8-2-1> Inhibition of MLR by Human Anti-human AILIM MonoclonalAntibody

[1147] PRMI1640 medium containing 10% FCS was added to each well of a96-well microplate having U-shaped wells. A solution of human anti-humanAILIM monoclonal antibody or mouse anti-human AILIM monoclonal antibodySA12 was diluted with PRMI1640 medium containing 10% FCS to preparesolutions with various concentrations of the antibody. Diluted antibodysolutions were added to the wells (final concentration: 0, 0.31, 1.25, 5and 20 μg/ml). Subsequently, T cells (50 μl) were added to the wells.The plate was incubated at 37° C. for 1 hour in a CO₂ incubator (NAPCO).After the reaction was completed, PBMCs (50 μl) derived from a differentdonor were added to the wells to initiate MLR.

[1148] When MLR was conducted using an antibody other than humananti-human AILIM antibody (described above in (3) to (6)) as the testsubstance, T cells derived from a different donor were allowed to reactafter the incubation of PBMCs with the test substance.

[1149] On the fifth day of the culture, tritium-labeled thymidine(³H-Thymidine; 20 μl; 1 μCi/well) diluted with PRMI1640 mediumcontaining 10% FCS was added to each well. Cultivation was continued forone day. After the culture was completed, the cells were harvested usinga Cell Harvester (Packard). Radioactivity of ³H incorporated of thecells was measured in a β-counter (TOP COUNT; Packard) to analyze therate of T cell proliferation after the culture.

[1150] Results are shown in FIGS. 48 to 59.

[1151] <8-2-2> Inhibition of MLR by Human Anti-human AILIM MonoclonalAntibody in MLR System where CTLA4-mediated Signaling Pathway has beenPreviously Blocked

[1152] PRMI1640 medium containing 10% FCS was added to each well of a96-well microplate having U-shaped wells. A solution of human anti-humanAILIM monoclonal antibody or mouse anti-human AILIM monoclonal antibodySA12 was diluted with PRMI1640 medium containing 10% FCS to preparesolutions with various concentrations of the antibody. The dilutedantibody solutions were added to the wells (final concentration: 0,0.31, 1.25, 5 and 20 μg/ml). Subsequently, T cells (50 μl) were added tothe wells. The plate was incubated at 37° C. for 1 hour in a CO₂incubator (NAPCO).

[1153] In addition to the culture of the T cells, PBMCs (in RPMI1640medium containing 10% FCS) derived from other donors were culturedindependently after adding human CTLA4-IgFc to the PBMCs. Cultivationwas performed at 37° C. for 1 hour in a CO₂ incubator (NAPCO).Concentration of CTLA4-IgFc was adjusted to 20 μg/ml at the start of theMLR.

[1154] Subsequently, PBMCs (50 μl) were added to the T cell culturedescribed above to initiate MLR.

[1155] When MLR was conducted using an antibody other than humananti-human AILIM antibody (described above in (3) to (5)) as the testsubstance, T cells derived from a different donor were allowed to reactafter the incubation of PBMCs, which had been cultured in the presenceof CTLA4-IgFc, with the test substance.

[1156] On the fifth day of the culture, tritium-labeled thymidine(³H-Thymidine; 20 μl; 1 μCi/well) diluted with PRMI1640 mediumcontaining 10% FCS was added to each well. Cultivation was continued forone day. After the culture was completed, the cells were harvested byusing a Cell Harvester (Packard). Radioactivity of ³H incorporated ofthe cells was measured in a β-counter (TOP COUNT; Packard) to analyzethe rate of T cell proliferation after the culture.

[1157] Results are shown in FIGS. 60 to 69.

[1158] The results obtained from the two tests described above aresummarized as follows:

[1159] (1) CTLA4-IgFc blocks the CTLA-4-mediated signal transduction,and thereby inhibiting the allogenic MLR-induced proliferation of Tcell.

[1160] (2) Anti-CD80 antibody and anti-CD86 antibody inhibit the signaltransduction mediated by CD80/CD86, which is a ligand for CTLA4 andCD28, and thereby inhibiting the allogenic MLR-induced proliferation ofT cell.

[1161] (3) A monoclonal antibody against human AILIM, like CTLA4-IgFc,anti-CD80 antibody and anti-CD86 antibody, significantly inhibits theallogenic MLR-induced T cell proliferation associated with theAILIM-mediated signal transduction in an antibodyconcentration-dependent manner.

[1162] In other words, these results show that a tertiary pathwaymediated by AILIM and the ligand thereof in addition to the knownpathways mediated by CTLA4/CD80/CD86 and mediated by CD28/CD80/CD86exist as a costimulatory signaling pathways required for T cellactivation, as well as that the AILIM-mediated signaling pathway isinhibited by antibody against AILIM.

[1163] Furthermore, it raises the possibility that contribution ofAILIM-mediated pathway to the signal transduction may be comparable tothose of CTLA4/CD80/CD86-mediated pathway and CD28/CD80/CD86-mediatedpathway.

EXAMPLE 9

[1164] Activity of Human Anti-human AILIM Monoclonal Antibody to InduceAntibody-dependent Cellular Cytotoxicity (ADCC)

[1165] Biological activities caused by antibodies include induction ofantibody-dependent cellular cytotoxicity (ADCC). ADCC is a cytotoxicaction that requires the antibody in addition to effector cells andtarget cells, that induces damage on the target cells induced by theeffector cells such as lymphocyte, macrophage or polymorphonuclearleucocyte.

[1166] The activity of the anti-human AILIM monoclonal antibody of thepresent invention to induce ADCC was analyzed as follows:

[1167]⁵¹Cr (0.1 mCi/10⁶ cells; Amersham-Pharmacia) was added to theculture of human AILIM-overexpressing recombinant HPB-ALL cells preparedin the Example described above, and the mixture was incubated at 37° C.for 2 hours. The cells were washed 8 times with RPMI1640 medium. Theisotope-labeled cells obtained were used as target cells.

[1168] Control experiments were performed using wild-type human HPB-ALLcells labeled with the isotope as control cells in the same manner asdescribed above.

[1169] By using Lymphosepar I (IBL), PBMC fractions were separated fromperipheral blood collected from normal healthy persons. The resultinghuman PBMCs were used as effector cells.

[1170] The target cells (1×10⁴ cells/well; 25 μl/well) were plated oneach well of a 96-well microplate (Nunc) having U-shaped wells.Subsequently, any one of various concentrations of human anti-humanAILIM monoclonal antibodies diluted with RPMI1640 medium containing 5%FBS (0.0001-1.0 μg/ml; 25 μl/well), the medium alone (25 μl/well) or 1%Nonidet P-40 (25 μl/well; detergent having cell-lysing activity) wasadded to each well and the plate was incubated at room temperature for20 minutes.

[1171] Cultivation was carried out using anti-human CD3 monoclonalantibody OKT3 (ATCC CRL-8001) as a positive control antibody instead ofanti-human AILIM antibody in the same manner as described above.

[1172] Subsequently, the effector cells (E/T ratio=50; 1×10⁵ cells/well;50 μl/well) were added to each well and the plate was incubated at 37°C. for 16 hours under an atmosphere of 5% CO₂ in an incubator.

[1173] After cultivation, samples were centrifuged (at 1,500 rpm at 4°C. for 10 minutes). Resulting supernatant was recovered. Radioactivityin the centrifugal supernatant was measured by a γ-counter. Theradioactivity represents the amount of ⁵¹Cr released from the cells intothe culture supernatant by the damage of cell membrane by ADCC.

[1174] Percentage of cell membrane damage (percentage cell lysis), whichwas caused by ADCC induced by anti-AILIM antibody or anti-CD3 antibody,was determined under an assumption that the radioactivity observed withthe medium alone corresponds to 0% with respect to the cell membranedamage (0) and that with Nonidet corresponds to 100% with respect to thecell membrane damage.

[1175] Results are shown in FIGS. 70 and 71.

[1176] The result of the test showed that human anti-human AILIMmonoclonal antibody of the present invention exhibited ADCC-inducingactivity in a concentration-dependent manner.

EXAMPLE 10

[1177] Determination of Gene and Amino Acid Sequences of HumanAnti-human AILIM Monoclonal Antibody and Analysis of the Same

[1178] Sequences of cDNAs encoding the heavy chains as well as cDNAsencoding the light chains of various human anti-human AILIM monoclonalantibodies, which had been prepared in the Example described above, weredetermined as described below. Structural features of the genes werealso analyzed.

[1179] By using Quick Prep mRNA Purification Kit (Amersham-Pharmacia),PolyA⁺RNAs were extracted and purified from each of hybridomas (clones:AIH5D3 (JMab-136), AII289 (JMab-138) and AII394 (JMab-139)), whichproduce human monoclonal antibody against human AILIM prepared in theExample described above.

[1180] The hybridoma cells were suspended in a cell lysis buffer (LysisBuffer), and lysed by using a syringe to solubilize them. Oligo (dT)resin was added to the solubilized material and the mixture was shakengently. Subsequently, Oligo (dT) resin was washed, and then PolyA⁺RNAwas eluted with Elution Buffer. Eluted PolyA⁺RNA was precipitated withethanol, and then dissolved in Tris-EDTA buffer. Concentration ofPolyA+RNA obtained was determined by absorbance at a wavelength of 260nm.

[1181] Double-stranded cDNA was synthesized by using PolyA⁺RNA as atemplate according to M-MLV Reverse Transcriptase method using acommercially available cDNA synthesis kit (GIBCOBRL) and synthetic oligoDNA NotI-T (SEQ ID NO:1) as a primer.

[1182] Specifically, single-stranded cDNA was synthesized in a solution(about 50 μl) containing PolyA⁺RNA (about 5 μg) purified from thehybridomas as a template, the primer (about 400 pmole) and M-MLV ReverseTranscriptase at 37° C. for 1 hour. Subsequently, dNTP, DNA polymeraseI, RNaseH, DNA ligase, buffer and distilled water were added to thereaction solution (4 μl), and the mixture was incubated at 16° C. for 2hours to synthesize double-stranded cDNA. The resulting double-strandedcDNA was extracted with phenol/chloroform and then precipitated withethanol.

[1183] Subsequently, EcoRI linker DNA (about 300 pmole) and DNA ligase(Ligation High; 33 μl; TOYOBO) was added to the solution containing thedouble-stranded cDNA in TE buffer (about 50 μl) and the mixture wasincubated at 16° C. for about 80 minutes to ligate the cDNA with thelinker DNA. The linker DNA used was a double-stranded DNA consisting ofoligo DNA (20adp; SEQ ID NO:2) and oligo DNA (24adp; SEQ ID NO:3), whichhad been 5′-phosphorylated and annealed to each other by a commonly usedmethod.

[1184] The DNA ligate was extracted with phenol/chloroform, and thenprecipitated with ethanol. Subsequently, the DNA reactant was digestedwith a commercially available restriction enzyme NotI (TOYOBO), and thenincubated with a commercially available ATP solution (GIBCO BRL) and T4kinase (TOYOBO) at 37° C. for 30 minutes to phosphorylate the 5′ endthereof.

[1185] The resulting DNA was precipitated with ethanol-, and thenfractionated by polyacrylamide gel electrophoresis. A piece of gelcontaining DNA of about 500 bp to2000 bp was cut out. Cutting of the gelwas carried out while the DNA stained with ethidium bromide was beingvisualized by irradiating UV light in a photographic device.

[1186] The gel cut off was crushed and then suspended in TE buffer. Thesuspension was centrifuged and the resulting supernatant was recovered.

[1187] The DNA recovered was ligated to a commercially available lambdaphage vector λEXcell (0.25 μg; Amersham Pharmacia) in the presence ofcommercially available DNA Ligase (Ligation High; TOYOBO) (at 16° C. for30 minutes). In the next step, the DNA ligate was packaged into lambdaphage using a commercially available lambda phage packaging kit GigapackIII Gold (STRATAGENE) and the resulting phage particles were infected toE. coli NM522 as a host to prepare a cDNA library. All the manipulationswere carried out according to the experimental protocol attached to thekit.

[1188] Subsequently, the cDNA library was screened by a plaquehybridization method (Maniatis et al., “Molecular Cloning: A LabolatoryManual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) asfollows:

[1189] The cDNA library (1×10⁴ plaques) was plated on agar plates andreplica filters thereof were prepared by using Hybond-N nylon membranes(Amersham Pharmacia). These replica filters were subjected tohybridization treatment using probes labeled by using γ³²P-ATP in ahybridization buffer according to the plaque hybridization method.Probes used were HIGLC (SEQ ID NO:4) for antibody light chain and NHCc2(SEQ ID NO:5) for antibody heavy chain. Single-plaque isolation wascarried out from the positive clones obtained in the primary screeningand secondary screening.

[1190] Each of heavy chain and light chain of the antibody was amplifiedby PCR using a single PCR primer and Taq PCR kit (TAKARA) by utilizingphage suspension from each positive clone as a template DNA. A pair ofprimers used for antibody light chain were ExcellE (SEQ ID NO:6) andck117 (SEQ ID NO:7), and a pair of primers used for antibody heavy chainwere ExcellE (SEQ ID NO:6) and NHCc2 (SEQ ID NO:5). The resulting PCRproducts were fractionated according to a usual method using agarose gelelectrophoresis. Pieces of gel containing DNAs of about 600 bpcorresponding to the heavy chain and light chain were cut out.Nucleotide sequences of the DNAs purified from the gel were analyzed byusing a DNA Sequencer (373A; PE-Applied Biosystems), ABI PRISMSequencing Software (PE-Applied Biosystems) and ABI PRISM Auto Assembler(PE-Applied Biosystems). DNA from each positive clone was verified tohave sufficient length of nucleotide sequence.

[1191] λPhage from the plaque of each positive clone was infected to E.coli NP66 for in vivo excision of plasmid DNA of interest, and theresulting filamentous phages were plated on ampicillin-containing platesto give colonies. Subsequently, plasmid DNAs were recovered and purifiedfrom the colonies by a commonly used method, and E. coli JM109 wastransformed with the plasmids. Subsequently, the transformed cells wereplated on ampicillin-containing nutrient agar plates to form colonies.

[1192] Subsequently, bacterial suspension in ampicillin-containing LBmedium derived from each colony was transferred to a liquid nutrientmedium and the bacteria were cultured at 37° C. for 24 hours. Thebacteria were harvested from the culture, and then the plasmid DNA waspurified by a plasmid purification kit (Quiagen). Each of the plasmidDNAs was digested with restriction enzymes EcoRI/NotI to verify thepresence of vector DNA and insert DNA (heavy chain cDNA or light chaincDNA).

[1193] Each nucleotide sequence of cDNA encoding heavy chain andantibody light chain of the antibody, which was inserted in eachpurified plasmid, was determined by a commonly used method using DNASequencer (377A; PE-Applied Biosystems), ABI PRISM Sequencing Software(PE-Applied Biosystems) and ABI PRISM Auto Assembler (PE-AppliedBiosystems).

[1194] Primers used for the sequence determination were as follows:

[1195] <Primers Used for the Determination of Heavy Chain cDNA>

[1196] M13R primer (SEQ ID NO:8; STRATAGENE), ExcellE (SEQ ID NO:6),136H (SEQ ID NO:9), 138/9H (SEQ ID NO:10), AILIMHC1 (SEQ ID NO:11), HCc1(SEQ ID NO:12), NHCc2 (SEQ ID NO:5), HCc7 (SEQ ID NO:13), HCc8 (SEQ IDNO:14), HCc3 (SEQ ID NO:15), HCc4 (SEQ ID NO:16), HCc6 (SEQ ID NO:17),HIGHC (SEQ ID NO:18), HCc9 (SEQ ID NO:19), HCc5 (SEQ ID NO:20) and polyA(SEQ ID NO:21).

[1197] <Primers Used for the Determination of Light Chain cDNA>

[1198] M13R primer (SEQ ID NO:8; STRATAGENE), ExcellE (SEQ ID NO:6),AILIMLC1 (SEQ ID NO:22), AILIMLC2 (SEQ ID NO:23), LCcl (SEQ ID NO:24),ck117 (SEQ ID NO:7), HIGLC (SEQ ID NO:4), LCc2 (SEQ ID NO:25), HIK (SEQID NO:26), and polyA (SEQ ID NO:21).

[1199] Sequence Listing shown below contains cDNA sequence encodingheavy chain and cDNA sequence encoding light chain of human monoclonalantibody against human AILIM, which are produced by each hybridomamentioned above, as well as amino acid sequences deduced from the cDNAsequences.

[1200] Clone AIH5D3 (JMab-136)

[1201] <Heavy Chain>

[1202] DNA sequence: SEQ ID NO:27 (signal sequence: nucleotide number 69to 125, V region: nucleotide number 126 to 419)

[1203] Amino acid sequence: SEQ ID NO:28 (comprising signal sequence:amino acid number 1 to 19, variable region: amino acid number 20 to 118)

[1204] <Light Chain>

[1205] DNA sequence: SEQ ID NO:29 (signal sequence: nucleotide number 39to 104, V region: nucleotide number 105 to 386)

[1206] Amino acid sequence: SEQ ID NO:30 (comprising signal sequence:amino acid number 1 to 22, variable region: amino acid number 23 to 116)

[1207] Clone AII289 (JMab-138)

[1208] <Heavy Chain>

[1209] DNA sequence: SEQ ID NO:31 (comprising signal sequence:nucleotide number 94 to 150, V region: nucleotide number 151 to 441)

[1210] Amino acid sequence: SEQ ID NO:32 (comprising signal sequence:amino acid number 1 to 19, variable region: amino acid number 20 to 116)

[1211] <Light Chain>

[1212] DNA sequence: SEQ ID NO:33 (comprising signal sequence:nucleotide number 28 to 87, V region: nucleotide number 88 to 375)

[1213] Amino acid sequence: SEQ ID NO:34 (comprising signal sequence:amino acid number 1 to 20, variable region: amino acid number 21 to 116)

[1214] Clone AII394 (JMab-139)

[1215] <Heavy Chain>

[1216] DNA sequence: SEQ ID NO:35 (signal sequence: nucleotide number 96to 152, V region: nucleotide number 153 to 443)

[1217] Amino acid sequence: SEQ ID NO:36 (comprising signal sequence:amino acid number 1 to 19, variable region: amino acid number 20 to 116)

[1218] <Light Chain>

[1219] DNA sequence: SEQ ID NO:37 (signal sequence: nucleotide number 33to 92, V region: nucleotide number 93 to 380)

[1220] Amino acid sequence: SEQ ID NO:38 (comprising signal sequence:amino acid number 1 to 20, variable region: amino acid number 21 to 116)

[1221] By using analytical software for gene sequence, the library VBASE Sequence for human immunoglobulin variable region genes constructedby Tomlinson et al. (Immunol. Today, Vol.16, No.5, p.237-242, 1995) wassearched for each of the DNA sequences determined herein.

[1222] Result showed that the V-region genes of the respective heavychain and light chain of the above-mentioned human monoclonal antibodiesconsisted of the following segments.

[1223] <Heavy chain V-region gene>

[1224] clone AIH5D3 (JMab-136): 1-02

[1225] clone AII289 (JMab-138): 3-13

[1226] clone AII394 (JMab-139): 3-13

[1227] <Light chain V-region gene>

[1228] clone AIH5D3 (JMab-136): L5

[1229] clone AII289 (JMab-138): A27

[1230] clone AII394 (JMab-139): A27

EXAMPLE 11

[1231] Inhibitory Effect of Human Anti-human AILIM Monoclonal Antibodyon Delayed-type Hypersensitivity (DTH)

[1232] The biological system of immune response, the function of whichis to eliminate harmful antigens (pathogenic microorganisms such asvirus, bacterium and parasite, foreign body, etc.) to the living bodies,and can be broadly divided into congenital immunity and acquiredimmunity.

[1233] The former is a system of elimination based on non-specificrecognition including phagocytosis by phagocytes (polymorphonuclearleukocyte, monocyte, macrophage, etc.), attack by natural killer (NK)cells, and opsonization of antigen by complement, etc.

[1234] The latter, acquired immune response, is a system of eliminationby lymphocytes (mainly, T cell and B cell) which acquired specificity(activation) to the antigen.

[1235] Further, acquired immune response can broadly be divided intocellular immunity and humoral immunity.

[1236] Unlike antibody-dependent humoral immunity, cellular immunity isan immune response expressed in general by the direct action of T cellon an antigen as the target. Cellular immunity is known to be involvedin immune response to virus or tumor, immune response induced aftertransplantation of tissue or organ, hypersensitivity to some drugs, andsome of autoimmune diseases.

[1237] Most typical phenomenon belonging to cellular immunity is thewell-known tuberculin allergy (almost synonymous with tuberculinhypersensitivity). Tuberculin allergy is a delayed allergy triggered bythe infection by tubercle bacillus. The allergy is due to the infectionby tubercle bacillus and can be induced by causing immune response byintracutaneously injecting, to a living body, tuberculin proteinproduced in culture supernatant of tubercle bacillus.

[1238] Delayed allergy is an allergy mediated by T cell (memory T cellmemorizing antigen) sensitized with an antigen. The allergy is called“delayed type,” because it takes 24 to 48 hours for a living bodysensitized with the antigen to express allergic response withinflammation induced by the memory T cell when contacted again with theantigen.

[1239] The phenomenon of tuberculin allergy, which is a representativeof delayed allergies, has generally been utilized in “tuberculin test”to diagnose whether or not sensitization by the infection of tuberclebacillus has already been established in an animal. Specifically, thetest is conducted as follows: purified tuberculin (purified proteinderivative; PPD; 0.1 ml of the derivative of 0.05 μg/0.1 ml (2.5 TU) forgeneral diagnostic use), which is tuberculin protein purified from theculture of tubercle bacillus, is intracutaneously given to an animal;the major axis of skin redness at the injection site is measured 48hours after the injection; and the presence of infection of tuberclebacillus can be diagnosed based on the major axis measured. If the majoraxis of the redness is shorter than 4 mm, then the subject is negative;if it is within the range of 4-9 mm then the subject is false positive;and if it is 10 mm or longer then the subject is decided positive.

[1240] Delayed allergies associated with cellular immunity include, forexample, Jones-Mote type hypersensitivity transiently induced by a smallamount of protein or the like, contact allergy to drugs such as picrylchloride or plant toxins such as Japanese lacquer, or allergy associatedwith graft rejection (e.g., allogenic graft) as well as theabove-mentioned allergy to antigen from infectious pathogen such astuberculin allergy caused by tubercle bacillus described above.

[1241] In this test, the inhibitory effect of anti-AILIM antibody ondelayed-type hypersensitivity (delayed allergy) was evaluated byutilizing the above-mentioned tuberculin test. The test was conducted asfollows:

[1242] Each of cynomolgus monkeys (male, body weight: 6.0-8.5 kg,Environmental Biological Life Science Research Center Inc.; 3individuals in each group), which had been sensitized with BCG (Bacillede Calmette et Guerin), which is an attenuated live bacterium ofbovine-type tubercle bacillus, was anesthetized with ketaminehydrochloride (10 mg/kg, intramuscular injection), and then any one ofthe test samples indicated below was intracutaneously given with aquantity of 0.1 ml to each injection site (6 injection sites/individual)in the chest. The distances between the injection sites of the samplewere 3 cm or longer.

[1243] (1) 1:1 mixed solution of human anti-human AILIM monoclonalantibody (JMab-136; 0.2 mg; 10 μg at each injection site) and tuberculin(4 μg/1 ml of physiological saline);

[1244] (2) 1:1 mixed solution of human anti-human AILIM monoclonalantibody (JMab-136; 2 mg; 100 μg at each injection site) and tuberculin(4 μg/1 ml of physiological saline);

[1245] (3) phosphate buffer (PBS (−)) as a control;

[1246] (4) 1:1 mixed solution of a commercially available steroidalanti-inflammatory agent, Prednisolone (0.2 mg; 10 μg at each injectionsite) and tuberculin (4 μg/1 ml of physiological saline) as a positivecontrol;

[1247] (5) 1:1 mixed solution of human anti-KLH monoclonal antibody(Example <2-2>; 0.2 mg; 10 μg at each injection site) and tuberculin (4μg/1 ml of physiological saline) as a negative control.

[1248] 24 hours after injection of each sample, the major axis and minoraxis of redness at each injection site were measured to determine thearea of the redness.

[1249] The result is shown in FIG. 72.

[1250] The result showed that redness due to delayed allergy wassignificantly inhibited in any groups subjected to injection of theanti-AILIM antibodies, as compared with the control and negative controlgroups. The inhibitory effect was comparable to that of the steroidalanti-inflammatory drug used as a positive control.

EXAMPLE 12

[1251] Analysis for the Expression of AILIM in Various Tissues ofPatients with Graft Versus Host Disease (GVHD)

[1252] Expression of AILIM in a variety of tissues obtained of biopsyfrom patients, who were recipients subjected to transplantation ofallogenic graft from donors and had been diagnosed clinically to beaffected with acute or chronic graft versus host disease (GVHD) afterthe transplantation, was analyzed by a commonly used method. Tissueswere stained with HE and human anti-human AILIM monoclonal antibody(JMab-36) prepared in the Example described above.

[1253] Analysis was carried out using 33 samples form various tissuescollected from acute GVHD patients (28 cases) as well as 5 samples fromchronic GVHD patient (5 cases).

[1254] Results were as follows: AILIM-positive reaction was found in 15of 29 samples of skin tissue; in 1 of 3 samples of stomach tissue; andin 1 of 1 sample of colon tissue; which were all obtained from acuteGVHD patients. AILIM-positive reaction was found in 1 of 3 samples ofskin tissue; in 2 of 2 samples of colon tissue; which were all obtainedfrom chronic GVHD patients. Further, AILIM-positive reaction was foundin 10 of 13 samples in which significant lymphocyte infiltration hadbeen observed.

EXAMPLE 13

[1255] Activity of Human Anti-human AILIM Monoclonal Antibody toTransduce Costimulatory Signal in Monkey T Cell

[1256] The experiment conducted in Example 7 has demonstrated that thehuman anti-human AILIM monoclonal antibodies of the present inventionare capable of enhancing the proliferation of human T cell viacontrolling the human T cell response, specifically, controlling thetransduction of AILIM-mediated costimulatory signal to the cell. In thistest, it was analyzed whether or not the human monoclonal antibodiesexhibit activity of enhancing cell proliferation of monkey T cell by thesame method as described in Example 7.

[1257] <13-1> Dilution of Antibody

[1258] Anti-human CD3 monoclonal antibody SP34 (BD-Pharmingen) wasdiluted with phosphate buffer (PBS) to a final concentration of 1 μg/ml.

[1259] The above-prepared human anti-human AILIM monoclonal antibodyJMAb136 was diluted with PBS to a final concentration of 40 μg/ml. Theantibody solutions were further diluted with PBS to prepare variousconcentrations of antibodies (40 μg/ml-0.064 μg/ml).

[1260] <13-2> Coating of Microplate with Antibody

[1261] Each well of 96-well microplates was coated with anti-human CD3monoclonal antibody SP34 (1 μg/ml; 25 μl to each well) and the humananti-human AILIM monoclonal antibody JMAbl36 (40 μg/ml-0.064 μg/ml; 25μl to each well). The plates were incubated at 37° C. for 2 hours.Subsequently, the antibody solutions were discarded, and each well waswashed 3 times with PBS. After the wash, RPMI1640 medium containing 10%FCS was added to each well (100 μl/well), and the plates were incubatedat 37° C. for 1 hour. Thus respective wells of the plates were coatedwith the antibodies mentioned above.

[1262] Control experiments were carried out in the same manner usingplates coated with human anti-KLH monoclonal antibody (JMab23; see theprevious Examples) as control antibodies instead of the human anti-humanAILIM monoclonal antibodies.

[1263] The microplates coated with the antibodies were used in thefollowing assays.

[1264] <13-3> Preparation of Monkey T Cell Suspension

[1265] Peripheral blood was collected from cynomolgus monkeys andmononuclear cells were fractionated by density gradient centrifugationusing NycoPrep1.077A (Nycomed). According to the experimental manual,monkey T cells were separated from the cynomolgus monkey mononuclearcells by using anti-human CD4 antibody M-T477 (BD-Pharmingen),anti-human CD8 antibody RPA-T8 (BD-Pharmingen), anti-mouse IgG microbead(Miltenyi) and a Magnetic Sorter. T cell count was determined using ahemacytometer. Monkey T cells were suspended in RPMI1640 mediumcontaining 10% FCS. Thus monkey T cell suspension (1×10⁶ cell/ml) wasprepared.

[1266] <13-4> Cell Culture

[1267] (1) Culture Using Microplate Coated with Anti-human CD3 Antibodyand Anti-human AILIM Antibody

[1268] Simian T cell suspension was added to each well of a microplatecoated with the antibody mentioned above and the plate was incubated at37° C. for 2 days in a CO₂ incubator.

[1269] After the culture was completed, respective microplates were usedin the following assays:

[1270] <13-5>Determination of Proliferation Activity of T Cell

[1271] Methyl [³H]thymidine (0.5 μCi/well; Amersham-Pharmacia) was addedto each well of the plates after incubation, and the plates wereincubated at 37° C. for 6 hours in a CO₂ incubator. After incubation,the cells were trapped on GF/C filters (Packard) by using a CellHarvester. Subsequently, the filters were dried at 40° C. for 3 hours orlonger, and then Microscinti 0 (20 μl/well; Packard) was added thereto.Radioactivity of 3H incorporated in the cells trapped on the filters wasmeasured by a β-counter (TOP COUNT) to analyze the degree of T cellproliferation after the culture.

[1272] The result is shown in FIG. 73.

[1273] The result of this assay showed that simian T cells weresignificantly proliferated depending on the concentration of the cellswhen microplates were coated with anti-human AILIM monoclonal antibody(human monoclonal antibody or mouse monoclonal antibody) together withanti-human CD3 antibody.

[1274] The result also suggests that the human anti-human AILIMmonoclonal antibodies of the present invention can bind to monkey AILIMand have the activity to regulate the function of monkey AILIM.

EXAMPLE 14

[1275] Establishment of Method for Identifying and QuantifyingSubstances Capable of Binding to AILIM or AILIM Ligand

[1276] A method of ELISA (Enzyme-linked Immuno solvent assay) wasestablished to identify or quantify a substance capable of binding toAILIM (ICOS) or AILIM ligand (B7h/B7RP1/GL50/LICOS).

[1277] The principle of the method described below in detail as anexample is based on estimating, by ELISA, the degree of inhibition onthe binding between soluble human AILIM (hAILIM-IgFc) and soluble humanAILIM ligand (hB7h-IgFc) caused by the substance.

[1278] <14-1> Sample

[1279] The following samples were used:

[1280] (1) Streptavidin-HRP (Southern Biotechnology Associates, Inc.);

[1281] (2) Soluble human AILIM ligand (fusion protein between theextracellular region of human B7h and the constant region of humanIgG1);

[1282] The protein was prepared by the method described below in <14-2>;

[1283] (3) Biotin-labeled soluble AILIM-IgFc;

[1284] The AILIM-IgFc was prepared by further purifying the antigenobtained according to the same method as described in earlierapplications (JP-A 11-29599 (Example 16 (2)) and WO98/3 8216 (Example 16(2))) of one of the present inventors, Tezuka;

[1285] (4) Human anti-human AILIM monoclonal antibody (JMab-136;described above);

[1286] (5) Human anti-KLH monoclonal antibody (negative controlantibody; JMab-23; described above);

[1287] (6) Phosphate buffer (PBS (−); Nikken Seibutsu);

[1288] (7) PRMI1640 medium (Nikken Seibutsu);

[1289] (8) Fetal calf serum (FCS; JRH-Bioscience);

[1290] (9) 30% Bovine serum albumin (BSA; Sigma);

[1291] (10) Tween20 (BioRad);

[1292] (11) TMB⁺ substrate chromogen (Dako).

[1293] <14-2> Preparation of Soluble Human AILIM Ligand (Fusion Protein(hB7h-IgFc) of the Extracellular Region of Human B7h and the ConstantRegion of Human IgG1)

[1294] Total RNA was prepared from human peripheral blood-derivedmononuclear cells in the same manner as described in the Example above.cDNA was synthesized from the obtained total RNA (5 μg) as a templateand by using Superscript Preamplification System for First Strand cDNASynthesis (GIBCO-BRL).

[1295] Subsequently, 5′primer(5′-GAGGTCTCCGCCCTCGAGATGCGGCTGGGCAGTCC-3′, SEQ ID NO:39) having XhoIrestriction site and 3′primer (5′-CACAGGACAGCCAGGGGATCCCACGTGGCCGCG-3′,SEQ ID NO:40) having BamHI restriction site at their respective endswere designed and synthesized to amplify cDNA encoding the extracellularregion of human AILIM ligand (hB7h) by PCR. By using the cDNA as atemplate and the primer pair, PCR was conducted to prepare a DNA havingXhoI and BamHI at respective ends thereof containing cDNA encoding theextracellular region of human B7h. The resulting PCR products weredigested with XhoI and BamHI, and fractioned by agarose gelelectrophoresis to isolate a band corresponding to about 720-bp cDNAfragment, that was predicted to encode the extracellular region ofinterest. The isolated cDNA fragment was subcloned into plasmidpBluescript II SK (+) (Stratagene) pre-digested with XhoI and BamHI. Itwas verified that the cDNA fragment contained the portion encoding theextracellular region of human B7h by sequencing analysis using anautomated fluorometric DNA sequencer (Applied Biosystems).

[1296] On the other hand, DNA encoding Fc of human IgG1 as a fusionpartner was prepared as a BamHI-XbaI DNA fragment (about 1.3 kb) bydigesting a plasmid (see, Cell, Vol.61, p.1303-1313, 1990; prepared byDr. Seed et al., at the Massachusetts General Hospital) with BamHI andXbaI. This fragment contained exons encoding the hinge regions of humanIgG1, Cγ12, and Cγ13.

[1297] The XhoI-BamHI fragment encoding the extracellular region ofhuman B7h (hB7h), and the BamHI-XbaI fragment containing exons encodingFc (abbreviated as “IgFc”) of human IgGI, prepared as described above,were subcloned into a plasmid pBluescript II SK (+) (Stratagene)pre-digested with XhoI and XbaI.

[1298] Subsequently, the plasmid was digested with XhoI and XbaI toprepare a DNA fragment about 1.8-kb containing fusion DNA between theextracellular region of human B7h and human IgFc. By using T4 DNAligase, this fusion DNA fragment was inserted into an expression vectorpME18S (Medical Immunology, Vol.20, No.1, p.27-32, 1990, and “Handbookfor Genetic Engineering,” Experimental Medicine, supplement, Yodosha,pp. 101-107, 1992) between XhoI and XbaI sites, to construct a plasmidphB7h-IgFc.

[1299] Monolayer COS7 cells (ATCC CRL-1651) cultured to be sub-confluentin DMEM medium containing 10% fetal calf serum and ampicillin, weretransformed with the plasmid phB7h-IgFc by electroporation to yieldtransformed cells.

[1300] The transformed cells were allowed to expresshB7h-IgFc byculturing them in serum-free ASF104 medium for 72 hours.

[1301] HB7h-IgFc was purified by using a Protein G Sepharose affinitycolumn (Amersham Pharmacia) as follows:

[1302] The above-mentioned culture supernatant was centrifuged to obtaincentrifugal supernatant. The resulting supernatant was loaded onto aProtein G Sepharose affinity column pre-equilibrated with a bindingbuffer. Subsequently, the column was washed with the binding buffer, andthen elution was performed with an elution buffer. The eluted solutionwas recovered and then dialyzed against phosphate buffer. The outerdialyzing buffer was changed twice or more. Thus purified hB7h-IgFc wasobtained.

[1303] <14-3> Dilution of Antibody and Soluble Human AILIM (□AILIM-IgFc)and Reaction Thereof

[1304] Original solutions (20 μg/ml) of anti-human AILIM monoclonalantibody (JMab-136) and human anti-KLH monoclonal antibody (JMab-23) asa negative control antibody were diluted in a series of 11 levels, andeach sample (200 μl) was combined and mixed well with 200 μl of RPMI1640medium containing 10% FCS. Thus various concentrations of antibodysolutions were prepared. Biotin-labeled hAILIM-IgFc (2 μl/tube; finalconcentration 1 μg/ml) was added to each of the prepared solutions withvarious concentrations. The resulting solutions were mixed well andincubated at room temperature for 30 minutes.

[1305] <14-4> Assay for the Activity of Anti-AILIM Monoclonal Antibodyto Inhibit the Binding Between hAILIM-IgFc and hB7h-IgFc

[1306] hB7h-IgFc was added to each well of a 96-well microplate (50μl/well (800 ng/well)). The plate was sealed and then incubated at 37°C. for 1 hour. Solution was removed from each well, and the wells werewashed 3 times with PBS (120 μl). Subsequently, PBS containing 0.5% BSA(100 μl/well) was added to each well to block the unreacted sites. Theplate was sealed and incubated at 37° C. for 1 hour. After incubation,the solution was removed, and then the wells were washed 3 times withPBS (120 μl). Subsequently, each sample (50 μl/well) prepared in <14-3>was added to the wells. The plate was sealed and incubated at 37° C. for1 hour. Solution was removed from each well. The wells were washed 3times with RPMI1640 medium containing 10% FCS (120 μl) pre-heated at 37°C. Subsequently, PBS containing 3.7% formalin (100 μl/well) was added toeach well, and the plate was incubated on ice for 5 minutes. Solutionwas removed from each well, and the wells were washed 3 times with 0.1%Tween20 (120 μl). Subsequently, Streptavidin-HRP (50 μl/well) was addedto each well. The plate was sealed and incubated at room temperature for30 minutes. Solution was removed from each well, and the plate waswashed 3 times with PBS containing 0.1% Tween20 (120 μl). Subsequently,TMB⁺ substrate chromogen (50 μl/well) was added to each well and theplate was incubated at room temperature for 20 minutes. Subsequently, 2Nsulfuric acid (50 μl/well) was added to each well to stop the reaction.Absorbance of each well was measured at a wavelength of 450 mn by aTHERMO max (Molecular Devices).

[1307] Results are shown in FIGS. 74 to 76.

[1308] The results showed that anti-AILIM antibody had the activity ofinhibiting the binding between hAILIM-IgFc and hB7h-IgFc in adosage-dependent manner.

[1309] Accordingly, this Example indicates that an assay systemillustrated by the present method can be utilized to screen and identifysubstances capable of binding to AILIM or AILIM ligand (for example,antibody or synthetic low molecular weight compound).

EXAMPLE 15

[1310] Activity of Human Anti-human AILIM Monoclonal Antibody to Inhibitthe Proliferation of Human T Cell Associated with the Transduction ofAILIM-AILIM Ligand-mediated Costimulatory Signal

[1311] It was analyzed whether or not the anti-human AILIM monoclonalantibodies of the present invention had regulating activity on thetransduction of signal mediated by AILIM on the surface of human T cell,based on the measurement of inhibitory effect of the human anti-humanAILIM monoclonal antibody on cell proliferation induced by contactinghuman T cell with AILIM ligand (B7h/B7RP1I/GL50/LICOS).

[1312] <15-1> Dilution of Antibody

[1313] Anti-human CD3 monoclonal antibody OKT3 (ATCC CRL-8001) wasdiluted with phosphate buffer (PBS) to a final concentration of 8 μg/ml.

[1314] The soluble human AILIM ligand (hB7h-lgFc) prepared above wasdiluted with PBS to a final concentration of 40 μg/ml. The antibodysolutions were further diluted with PBS to prepare variousconcentrations of antibodies (40 μg/ml-0.064 μg/ml).

[1315] <15-2> Coating of Microplate with Antibody

[1316] Each well of 96-well microplates was coated with (1) anti-humanCD3 monoclonal antibody OKT3 (8 μg/ml; 25 μl in each well) and hB7h-lgFc(40 μg/ml-0.064 μg/ml; 25 μl in each well). The plates were incubated at37° C. for 2 hours. Subsequently, the antibody solutions were discarded,and each well was washed 3 times with PBS. After the wash, RPMI1640medium containing 10% FCS was added to each well (100 μl/well), and theplates were incubated at 37° C. for 1 hour. Thus respective wells of theplates were coated.

[1317] <15-3> Preparation of Human T Cell Suspension

[1318] Peripheral blood was collected from normal healthy persons andthe mononuclear cells were fractionated by density-gradientcentrifugation using LymphoPrep (Nycomed). According to the experimentalmanual, human T cells were separated from the human mononuclear cells byusing a Pan-T cell Isolation Kit (Miltenyi) and a Magnetic Sorter. Tcell count was determined by using a hemacytometer. Human T cells weresuspended in a RPMI1640 medium containing 10% FCS supplied with humananti-human AILIM monoclonal antibody JMab136 (20 μg/ml). Human T cellsuspension (1×10⁶ cells/ml) prepared was incubated at room temperaturefor 30 minutes.

[1319] Human anti-KLH monoclonal antibody JMAb23 (20 μg/ml) was used asa negative control antibody.

[1320] <15-4> Cell Culture

[1321] In the same manner as described above, human T cell suspension(100 μl/well; 1×10⁵ cells/well) was added to each well of a microplatecoated with anti-human CD3 antibody and hB7h-IgFc, and the plate wasincubated at 37° C. for 3 days in a CO₂ incubator.

[1322] <15-5> Determination of Proliferation Activity of T Cell

[1323] Methyl [³H]thymidine (0.5 μCi/well; Amersham-Pharmacia) was addedto each well of the plates after cultivation, and the plates wereincubated at 37° C. for 6 hours in a CO₂ incubator. After incubation,the cells were trapped on GF/C filters (Packard) by using a CellHarvester. Subsequently, the filters were dried at 40° C. for 3 hours orlonger, and then Microscinti 0 (20 μl/well; Packard) was added thereto.Radioactivity of ³H incorporated in the cells trapped on the filters wasmeasured by a β-counter (TOP COUNT) to analyze the degree of T cellproliferation after the culture.

[1324] The result is shown in FIG. 77.

[1325] The result obtained in this test showed that human T cells grewsignificantly depending on the concentration of human B7h-IgFc (in theassay using the negative control antibody). In addition, the anti-humanAILIM monoclonal antibody significantly inhibited the proliferation ofhuman T cells.

EXAMPLE 16

[1326] Activity of Human Anti-human AILIM Monoclonal Antibody to Inhibitthe Proliferation of Monkey T Cell Associated with the Transduction ofAILIM-AILIM Ligand-mediated Costimulatory Signal

[1327] The same test was conducted by using monkey T cells instead ofhuman T cells used in Example 15 described above.

[1328] <16-1> Dilution of Antibody and Others

[1329] Anti-human CD3 monoclonal antibody SP34 (BD-Pharmingen) wasdiluted with phosphate buffer (PBS) to a final concentration of 1 μg/ml.

[1330] The human B7h-IgFc prepared above was diluted with PBS to a finalconcentration of 40 μg/ml. The antibody solution was further dilutedwith PBS to prepare various concentrations of the antibody (40μg/ml-0.0064 μg/ml).

[1331] <16-2> Coating of Microplate with Antibody

[1332] Each well of 96-well microplates was coated with (1) anti-humanCD3 monoclonal antibody SP34 (BD-Pharmingen) (1 μg/ml; 25 μl in eachwell) and human B7h-IgFc (40 μg/ml-0.006 μg/ml; 25 μl in each well). Theplates were incubated at 37° C. for 2 hours. Subsequently, the antibodysolution was discarded, and each well was washed 3 times with PBS. Afterthe wash, RPMI1640 medium containing 10% FCS was added to each well (100μl/well), and the plates were incubated at 37° C. for 1 hour. Thus,respective wells of the plates were coated with the antibody.

[1333] <16-3> Preparation of Monkey T Cell Suspension

[1334] Peripheral blood was collected from cynomolgus monkeys. Afraction containing mononuclear cells was prepared by density-gradientcentrifugation using NycoPrep1.077A (Nycomed). According to theexperimental manual, monkey T cells were separated from the cynomolgusmonkey mononuclear cells by using anti-human CD4 antibody M-T477(BD-Pharmingen), anti-human CD8 antibody RPA-T8 (BD-Pharmingen),anti-mouse IgG microbead (Miltenyi) and a Magnetic Sorter. T cell countwas determined by using a hemacytometer. Monkey T cells were suspendedin RPMI1640 medium containing human anti-human AILIM monoclonal antibodyJMab-136 (20 μg/ml) and 10% FCS to prepare monkey T cell suspension(1×10⁶ cells/ml). The suspension was incubated at room temperature for30 minutes.

[1335] Human anti-KLH monoclonal antibody JMab-23 (20 μg/ml) was used asa negative control antibody.

[1336] <16-4> Cell Culture

[1337] In the same manner as described above, monkey T cell suspension(100 μl/well; 1×10⁵ cells/well) was added to each well of a microplatecoated with anti-human CD3 antibody and hB7h-IgFc, and the plate wasincubated at 37° C. for 3 days in a CO₂ incubator.

[1338] <16-5> Determination of Activity of T Cell Proliferation

[1339] Methyl [³H]thymidine (0.5 μCi/well; Amersham-Pharmacia) was addedto each well of the plates after the cultivation, and the plates wereincubated at 37° C. for 6 hours in a CO₂ incubator. After theincubation, the cells were trapped on GF/C filters (Packard) by using aCell Harvester. Subsequently, the filters were dried at 40° C. for 3hours or longer, and then Microscinti 0 (20 μl/well; Packard) was addedthereto. Radioactivity of ³H incorporated into the cells trapped on thefilters was measured by a b-counter (TOP COUNT) to analyze the degree ofT cell proliferation after the culture.

[1340] The result is shown in FIG. 78.

[1341] The result obtained in this test showed that monkey T cells grewsignificantly depending on the concentration of human B7h-IgFc (in theassay using the negative control antibody). In addition, the anti-humanAILIM monoclonal antibody significantly inhibited the proliferationmonkey T cells.

Sequence Listing Free Text

[1342] SEQ ID NO:1

[1343] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, NotI-T.

[1344] SEQ ID NO:2

[1345] Other Information: Description of Artificial Sequence:Artificially synthesized linker sequence, 20adp

[1346] SEQ ID NO:3

[1347] Other Information: Description of Artificial Sequence:Artificially synthesized linker sequence, 24adp.

[1348] SEQ ID NO:4

[1349] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HIGLC.

[1350] SEQ ID NO:5

[1351] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, NHCc2.

[1352] SEQ ID NO:6

[1353] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, ExcellE.

[1354] SEQ ID NO:7

[1355] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, ck117.

[1356] SEQ ID NO:8

[1357] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, M13R.

[1358] SEQ ID NO:9

[1359] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, 136H.

[1360] SEQ ID NO:10

[1361] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, 138/9H.

[1362] SEQ ID NO:11

[1363] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, AILIMHC1.

[1364] SEQ ID NO:12

[1365] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HCc1.

[1366] SEQ ID NO:13

[1367] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HCc7.

[1368] SEQ ID NO:14

[1369] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HCc8.

[1370] SEQ ID NO:15

[1371] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HCc3.

[1372] SEQ ID NO:16

[1373] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HCc4.

[1374] SEQ ID NO:17

[1375] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HCc6.

[1376] SEQ ID NO: 18

[1377] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HIGHC.

[1378] SEQ ID NO:19

[1379] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HCc9.

[1380] SEQ ID NO:20

[1381] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HCc5.

[1382] SEQ ID NO:21

[1383] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, polyA.

[1384] SEQ ID NO:22

[1385] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, AILIMLC1.

[1386] SEQ ID NO:23

[1387] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, AILIMLC2.

[1388] SEQ ID NO:24

[1389] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, LCc1.

[1390] SEQ ID NO:25

[1391] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, LCc2.

[1392] SEQ ID NO:26

[1393] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence, HIK.

[1394] SEQ ID NO:39

[1395] Other Information: Description of Artificial Sequence:Artificially synthesized primer sequence

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 43 <210> SEQ ID NO 1<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Artificially synthesized primersequence, NotI-T <221> NAME/KEY: primer_bind <222> LOCATION: (1)...(45)<400> SEQUENCE: 1 aactggaagc ttcagcggcc gcagagattt tttttttttt ttttt 45<210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Artificiallysynthesized linker sequence, 20adp <400> SEQUENCE: 2 cgtggtgtcatggcactgcg 20 <210> SEQ ID NO 3 <211> LENGTH: 24 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Artificially synthesized linker sequence, 24adp <400> SEQUENCE: 3aattcgcagt gccatgacac cacg 24 <210> SEQ ID NO 4 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HIGLC <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(23) <400> SEQUENCE: 4gtctgctttg ctcagcgtca ggg 23 <210> SEQ ID NO 5 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, NHCc2 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 5caccggttcg gggaagtagt c 21 <210> SEQ ID NO 6 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, ExcellE <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 6ggtgacacta tagaatacag g 21 <210> SEQ ID NO 7 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, ck117 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(25) <400> SEQUENCE: 7gcaggcacac aacagaggca gttcc 25 <210> SEQ ID NO 8 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, M13R <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(20) <400> SEQUENCE: 8cacaggaaac agctatgacc 20 <210> SEQ ID NO 9 <211> LENGTH: 21 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, 136H <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 9cctggacaag ggcttgagtg g 21 <210> SEQ ID NO 10 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, 138/9H <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 10acaggaaaag gtctggagtg g 21 <210> SEQ ID NO 11 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, AILIMHC1 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 11acagtaatac acggccgtgt c 21 <210> SEQ ID NO 12 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HCc1 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 12gactacttcc ccgaaccggt g 21 <210> SEQ ID NO 13 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HCc7 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(22) <400> SEQUENCE: 13gtggcaggac cgtcagtctt cc 22 <210> SEQ ID NO 14 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HCc8 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(24) <400> SEQUENCE: 14aagaggaaga ctgacggtcc tgcc 24 <210> SEQ ID NO 15 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HCc3 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(23) <400> SEQUENCE: 15ccgttgtgca ccaggactgg ctg 23 <210> SEQ ID NO 16 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HCc4 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(23) <400> SEQUENCE: 16tgcacttgta ctccttgccg ttc 23 <210> SEQ ID NO 17 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HCc6 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(23) <400> SEQUENCE: 17cagccggaga acaactacaa gac 23 <210> SEQ ID NO 18 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HIGHC <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(22) <400> SEQUENCE: 18tcttgtagtt gttctccggc tg 22 <210> SEQ ID NO 19 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HCc9 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(22) <400> SEQUENCE: 19tacttcccag gcacccagca tg 22 <210> SEQ ID NO 20 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HCc5 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(23) <400> SEQUENCE: 20atgctgggtg cctgggaagt atg 23 <210> SEQ ID NO 21 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, polyA <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 21tcaaactatc ggccttgctg g 21 <210> SEQ ID NO 22 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, AILIMLC1 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 22tagcctggta tcagcagaaa c 21 <210> SEQ ID NO 23 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, AILIMLC2 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 23gtttctgctg ataccaggct a 21 <210> SEQ ID NO 24 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, LCc1 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(22) <400> SEQUENCE: 24gcaccatctg tcttcatctt cc 22 <210> SEQ ID NO 25 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, LCc2 <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(21) <400> SEQUENCE: 25caaagagctt caacagggga g 21 <210> SEQ ID NO 26 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence, HIK <221>NAME/KEY: primer_bind <222> LOCATION: (1)...(20) <400> SEQUENCE: 26aggctggaac tgaggagcag 20 <210> SEQ ID NO 27 <211> LENGTH: 1616 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:5′UTR <222> LOCATION: (1)...(68) <221> NAME/KEY: CDS <222> LOCATION:(69)...(1478) <221> NAME/KEY: 3′UTR <222> LOCATION: (1482)...(1616)<221> NAME/KEY: sig_peptide <222> LOCATION: (69)...(125) <400> SEQUENCE:27 gaattcgcag tgccatgaca ccacgcatct gtcctctaga gaatcccctg agagctccgt 60tcctcacc atg gac tgg acc tgg agg atc ctc ttc ttg gtg gca gca gcc 110 MetAsp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala -15 -10 aca gga gcccac tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg 158 Thr Gly Ala HisSer Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val -5 1 5 10 aag aag cctggg gcc tca gtg aag gtc tcc tgc aag gct tct gga tac 206 Lys Lys Pro GlyAla Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 15 20 25 acc ttc acc ggctac tat atg cac tgg gtg cga cag gcc cct gga caa 254 Thr Phe Thr Gly TyrTyr Met His Trp Val Arg Gln Ala Pro Gly Gln 30 35 40 ggg ctt gag tgg atggga tgg atc aac cct cac agt ggt ggc aca aac 302 Gly Leu Glu Trp Met GlyTrp Ile Asn Pro His Ser Gly Gly Thr Asn 45 50 55 tat gca cag aag ttt cagggc agg gtc acc atg acc agg gac acg tcc 350 Tyr Ala Gln Lys Phe Gln GlyArg Val Thr Met Thr Arg Asp Thr Ser 60 65 70 75 atc agc aca gcc tac atggag ctg agc agg ctg aga tcc gac gac acg 398 Ile Ser Thr Ala Tyr Met GluLeu Ser Arg Leu Arg Ser Asp Asp Thr 80 85 90 gcc gtg tat tac tgt gcg aggacg tat tac tat gat agt agt ggt tat 446 Ala Val Tyr Tyr Cys Ala Arg ThrTyr Tyr Tyr Asp Ser Ser Gly Tyr 95 100 105 tac cat gat gct ttt gat atctgg ggc caa ggg aca atg gtc acc gtc 494 Tyr His Asp Ala Phe Asp Ile TrpGly Gln Gly Thr Met Val Thr Val 110 115 120 tct tca gcc tcc acc aag ggccca tcg gtc ttc ccc ctg gcg ccc tgc 542 Ser Ser Ala Ser Thr Lys Gly ProSer Val Phe Pro Leu Ala Pro Cys 125 130 135 tcc agg agc acc tcc gag agcaca gcg gcc ctg ggc tgc ctg gtc aag 590 Ser Arg Ser Thr Ser Glu Ser ThrAla Ala Leu Gly Cys Leu Val Lys 140 145 150 155 gac tac ttc ccc gaa ccggtg acg gtg tcg tgg aac tca ggc gct ctg 638 Asp Tyr Phe Pro Glu Pro ValThr Val Ser Trp Asn Ser Gly Ala Leu 160 165 170 acc agc ggc gtg cac accttc cca gct gtc cta cag tcc tca gga ctc 686 Thr Ser Gly Val His Thr PhePro Ala Val Leu Gln Ser Ser Gly Leu 175 180 185 tac tcc ctc agc agc gtggtg acc gtg ccc tcc agc aac ttc ggc acc 734 Tyr Ser Leu Ser Ser Val ValThr Val Pro Ser Ser Asn Phe Gly Thr 190 195 200 cag acc tac acc tgc aacgta gat cac aag ccc agc aac acc aag gtg 782 Gln Thr Tyr Thr Cys Asn ValAsp His Lys Pro Ser Asn Thr Lys Val 205 210 215 gac aag aca gtt gag cgcaaa tgt tgt gtc gag tgc cca ccg tgc cca 830 Asp Lys Thr Val Glu Arg LysCys Cys Val Glu Cys Pro Pro Cys Pro 220 225 230 235 gca cca cct gtg gcagga ccg tca gtc ttc ctc ttc ccc cca aaa ccc 878 Ala Pro Pro Val Ala GlyPro Ser Val Phe Leu Phe Pro Pro Lys Pro 240 245 250 aag gac acc ctc atgatc tcc cgg acc cct gag gtc acg tgc gtg gtg 926 Lys Asp Thr Leu Met IleSer Arg Thr Pro Glu Val Thr Cys Val Val 255 260 265 gtg gac gtg agc cacgaa gac ccc gag gtc cag ttc aac tgg tac gtg 974 Val Asp Val Ser His GluAsp Pro Glu Val Gln Phe Asn Trp Tyr Val 270 275 280 gac ggc gtg gag gtgcat aat gcc aag aca aag cca cgg gag gag cag 1022 Asp Gly Val Glu Val HisAsn Ala Lys Thr Lys Pro Arg Glu Glu Gln 285 290 295 ttc aac agc acg ttccgt gtg gtc agc gtc ctc acc gtt gtg cac cag 1070 Phe Asn Ser Thr Phe ArgVal Val Ser Val Leu Thr Val Val His Gln 300 305 310 315 gac tgg ctg aacggc aag gag tac aag tgc aag gtc tcc aac aaa ggc 1118 Asp Trp Leu Asn GlyLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 320 325 330 ctc cca gcc cccatc gag aaa acc atc tcc aaa acc aaa ggg cag ccc 1166 Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 335 340 345 cga gaa cca caggtg tac acc ctg ccc cca tcc cgg gag gag atg acc 1214 Arg Glu Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 350 355 360 aag aac cag gtcagc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc 1262 Lys Asn Gln Val SerLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 365 370 375 gac atc gcc gtggag tgg gag agc aat ggg cag ccg gag aac aac tac 1310 Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 380 385 390 395 aag acc acacct ccc atg ctg gac tcc gac ggc tcc ttc ttc ctc tac 1358 Lys Thr Thr ProPro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 400 405 410 agc aag ctcacc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc 1406 Ser Lys Leu ThrVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 415 420 425 tca tgc tccgtg atg cat gag gct ctg cac aac cac tac acg cag aag 1454 Ser Cys Ser ValMet His Glu Ala Leu His Asn His Tyr Thr Gln Lys 430 435 440 agc ctc tccctg tct ccg ggt aaa tgagtgccac ggccggcaag cccccgctcc 1508 Ser Leu SerLeu Ser Pro Gly Lys 445 450 ccaggctctc ggggtcgcgt gaggatgctt ggcacgtaccccgtgtacat acttcccagg 1568 cacccagcat ggaaataaag cacccagcgc tgccctggaaaaaaaaaa 1616 <210> SEQ ID NO 28 <211> LENGTH: 470 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 28 Met Asp Trp Thr Trp Arg IleLeu Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Gln Val GlnLeu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45 Thr Gly Tyr Tyr Met HisTrp Val Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Trp IleAsn Pro His Ser Gly Gly Thr Asn Tyr Ala 65 70 75 80 Gln Lys Phe Gln GlyArg Val Thr Met Thr Arg Asp Thr Ser Ile Ser 85 90 95 Thr Ala Tyr Met GluLeu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val 100 105 110 Tyr Tyr Cys AlaArg Thr Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr His 115 120 125 Asp Ala PheAsp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 130 135 140 Ala SerThr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr GlnThr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys ValAsp Lys 225 230 235 240 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro ProCys Pro Ala Pro 245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe ProPro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu ValThr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val GlnPhe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Phe Asn 305 310 315 320 Ser Thr Phe Arg Val ValSer Val Leu Thr Val Val His Gln Asp Trp 325 330 335 Leu Asn Gly Lys GluTyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350 Ala Pro Ile GluLys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410415 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> SEQ ID NO 29 <211>LENGTH: 974 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: 5′UTR <222> LOCATION: (1)...(38) <221> NAME/KEY: CDS<222> LOCATION: (39)...(746) <221> NAME/KEY: 3′UTR <222> LOCATION:(750)...(974) <221> NAME/KEY: sig_peptide <222> LOCATION: (39)...(104)<400> SEQUENCE: 29 gaattcgcag tgccatgaca ccacggtcag gacacagc atg gac atgagg gtc ccc 56 Met Asp Met Arg Val Pro -20 gct cag ctc ctg ggg ctc ctgctg ctc tgg ttc cca ggt tcc aga tgc 104 Ala Gln Leu Leu Gly Leu Leu LeuLeu Trp Phe Pro Gly Ser Arg Cys -15 -10 -5 gac atc cag atg acc cag tctcca tct tcc gtg tct gca tct gta gga 152 Asp Ile Gln Met Thr Gln Ser ProSer Ser Val Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgtcgg gcg agt cag ggt att agc agg ttg 200 Asp Arg Val Thr Ile Thr Cys ArgAla Ser Gln Gly Ile Ser Arg Leu 20 25 30 tta gcc tgg tat cag cag aaa ccaggg aaa gcc cct aaa ctc ctg atc 248 Leu Ala Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys Leu Leu Ile 35 40 45 tat gtt gca tcc agt ttg caa agt ggggtc cca tca agg ttc agc ggc 296 Tyr Val Ala Ser Ser Leu Gln Ser Gly ValPro Ser Arg Phe Ser Gly 50 55 60 agt gga tct ggg aca gat ttc act ctc accatc agc agc ctg cag cct 344 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr IleSer Ser Leu Gln Pro 65 70 75 80 gaa gat ttt gca act tac tat tgt caa caggct aac agt ttc ccg tgg 392 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln AlaAsn Ser Phe Pro Trp 85 90 95 acg ttc ggc caa ggg acc aag gtg gaa atc aaacga act gtg gct gca 440 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys ArgThr Val Ala Ala 100 105 110 cca tct gtc ttc atc ttc ccg cca tct gat gagcag ttg aaa tct gga 488 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu GlnLeu Lys Ser Gly 115 120 125 act gcc tct gtt gtg tgc ctg ctg aat aac ttctat ccc aga gag gcc 536 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe TyrPro Arg Glu Ala 130 135 140 aaa gta cag tgg aag gtg gat aac gcc ctc caatcg ggt aac tcc cag 584 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln SerGly Asn Ser Gln 145 150 155 160 gag agt gtc aca gag cag gac agc aag gacagc acc tac agc ctc agc 632 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp SerThr Tyr Ser Leu Ser 165 170 175 agc acc ctg acg ctg agc aaa gca gac tacgag aaa cac aaa gtc tac 680 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr GluLys His Lys Val Tyr 180 185 190 gcc tgc gaa gtc acc cat cag ggc ctg agctcg ccc gtc aca aag agc 728 Ala Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser 195 200 205 ttc aac agg gga gag tgt tagagggagaagtgccccca cctgctcctc 776 Phe Asn Arg Gly Glu Cys 210 agttccagcctgaccccctc ccatcctttg gcctctgacc ctttttccac aggggaccta 836 cccctattgcggtcctccag ctcatctttc acctcacccc cctcctcctc cttggcttta 896 attatgctaatgttggagga gaatgaataa ataaagtgaa tctttgcaaa aaaaaaaaaa 956 aaaaatctctgcggccgc 974 <210> SEQ ID NO 30 <211> LENGTH: 236 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 30 Met Asp Met Arg Val Pro AlaGln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg CysAsp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 Val Ser Ala Ser Val GlyAsp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Ser Arg LeuLeu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu IleTyr Val Ala Ser Ser Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu GlnPro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 Ala Asn Ser PhePro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg ThrVal Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu GlnLeu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly LeuSer 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230235 <210> SEQ ID NO 31 <211> LENGTH: 1708 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: 5′UTR <222>LOCATION: (1)...(93) <221> NAME/KEY: CDS <222> LOCATION: (94)...(1503)<221> NAME/KEY: 3′UTR <222> LOCATION: (1507)...(1708) <221> NAME/KEY:sig_peptide <222> LOCATION: (94)...(150) <221> NAME/KEY: misc_feature<222> LOCATION: (1)...(1708) <223> OTHER INFORMATION: n = A,T,C or G<400> SEQUENCE: 31 gaattcgcag taccatgaca ccacgggagc cccagccttgggattcccaa gtgtttgtaa 60 tcagtgatca ggactgagca cacaggactc acc atg gagttg ggg ctg agc tgg 114 Met Glu Leu Gly Leu Ser Trp -15 gtt ttc ctt gttgct ata tta gaa ggt gtc cag tgt gag gtg cag ctg 162 Val Phe Leu Val AlaIle Leu Glu Gly Val Gln Cys Glu Val Gln Leu -10 -5 1 gtg gag tct ggg ggaggc ttg gta cag cct ggg ggg tcc ctg aga ctc 210 Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly Gly Ser Leu Arg Leu 5 10 15 20 tcc tgt gca gcc tctgga ttc acc ttc agt agc tac gac atg cac tgg 258 Ser Cys Ala Ala Ser GlyPhe Thr Phe Ser Ser Tyr Asp Met His Trp 25 30 35 gtc cgc caa gct aca ggaaaa ggt ctg gag tgg gtc tca gct att ggt 306 Val Arg Gln Ala Thr Gly LysGly Leu Glu Trp Val Ser Ala Ile Gly 40 45 50 act gct ggt gac aca tac tatcca ggc tcc gtg aag ggc cga ttc acc 354 Thr Ala Gly Asp Thr Tyr Tyr ProGly Ser Val Lys Gly Arg Phe Thr 55 60 65 atc tcc aga gaa aat gcc aag aactcc ttg tat ctt caa atg aac agc 402 Ile Ser Arg Glu Asn Ala Lys Asn SerLeu Tyr Leu Gln Met Asn Ser 70 75 80 ctg aga gcc ggg gac acg gct gtg tattac tgt gta aga gat aat agg 450 Leu Arg Ala Gly Asp Thr Ala Val Tyr TyrCys Val Arg Asp Asn Arg 85 90 95 100 aag gtg acc cac gag cac tac tac tactac ggt atg gac gtc tgg ggc 498 Lys Val Thr His Glu His Tyr Tyr Tyr TyrGly Met Asp Val Trp Gly 105 110 115 caa ggg acc acg gtc acc gtc tcc tcagcc tcc acc aag ggc cca tcg 546 Gln Gly Thr Thr Val Thr Val Ser Ser AlaSer Thr Lys Gly Pro Ser 120 125 130 gtc ttc ccc ctg gcg ccc tgc tcc aggagc acc tcc gag agc aca gcg 594 Val Phe Pro Leu Ala Pro Cys Ser Arg SerThr Ser Glu Ser Thr Ala 135 140 145 gcc ctg ggc tgc ctg gtc aag gac tacttc ccc gaa ccg gtg acg gtg 642 Ala Leu Gly Cys Leu Val Lys Asp Tyr PhePro Glu Pro Val Thr Val 150 155 160 tcg tgg aac tca ggc gct ctg acc agcggc gtg cac acc ttc cca gct 690 Ser Trp Asn Ser Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala 165 170 175 180 gtc cta cag tcc tca gga ctc tactcc ctc agc agc gtg gtg acc gtg 738 Val Leu Gln Ser Ser Gly Leu Tyr SerLeu Ser Ser Val Val Thr Val 185 190 195 ccc tcc agc aac ttc ggc acc cagacc tac acc tgc aac gta gat cac 786 Pro Ser Ser Asn Phe Gly Thr Gln ThrTyr Thr Cys Asn Val Asp His 200 205 210 aag ccc agc aac acc aag gtg gacaag aca gtt gag cgc aaa tgt tgt 834 Lys Pro Ser Asn Thr Lys Val Asp LysThr Val Glu Arg Lys Cys Cys 215 220 225 gtc gag tgc cca ccg tgc cca gcacca cct gtg gca gga ccg tca gtc 882 Val Glu Cys Pro Pro Cys Pro Ala ProPro Val Ala Gly Pro Ser Val 230 235 240 ttc ctc ttc ccc cca aaa ccc aaggac acc ctc atg atc tcc cgg acc 930 Phe Leu Phe Pro Pro Lys Pro Lys AspThr Leu Met Ile Ser Arg Thr 245 250 255 260 cct gag gtc acg tgc gtg gtggtg gac gtg agc cac gaa gac ccc gag 978 Pro Glu Val Thr Cys Val Val ValAsp Val Ser His Glu Asp Pro Glu 265 270 275 gtc cag ttc aac tgg tac gtggac ggc gtg gag gtg cat aat gcc aag 1026 Val Gln Phe Asn Trp Tyr Val AspGly Val Glu Val His Asn Ala Lys 280 285 290 aca aag cca cgg gag gag cagttc aac agc acg ttc cgt gtg gtc agc 1074 Thr Lys Pro Arg Glu Glu Gln PheAsn Ser Thr Phe Arg Val Val Ser 295 300 305 gtc ctc acc gtt gtg cac caggac tgg ctg aac ggc aag gag tac aag 1122 Val Leu Thr Val Val His Gln AspTrp Leu Asn Gly Lys Glu Tyr Lys 310 315 320 tgc aag gtc tcc aac aaa ggcctc cca gcc ccc atc gag aaa acc atc 1170 Cys Lys Val Ser Asn Lys Gly LeuPro Ala Pro Ile Glu Lys Thr Ile 325 330 335 340 tcc aaa acc aaa ggg cagccc cga gaa cca cag gtg tac acc ctg ccc 1218 Ser Lys Thr Lys Gly Gln ProArg Glu Pro Gln Val Tyr Thr Leu Pro 345 350 355 cca tcc cgg gag gag atgacc aag aac cag gtc agc ctg acc tgc ctg 1266 Pro Ser Arg Glu Glu Met ThrLys Asn Gln Val Ser Leu Thr Cys Leu 360 365 370 gtc aaa ggc ttc tac cccagc gac atc gcc gtg gag tgg gag agc aat 1314 Val Lys Gly Phe Tyr Pro SerAsp Ile Ala Val Glu Trp Glu Ser Asn 375 380 385 ggg cag ccg gag aac aactac aag acc aca cct ccc atg ctg gac tcc 1362 Gly Gln Pro Glu Asn Asn TyrLys Thr Thr Pro Pro Met Leu Asp Ser 390 395 400 gac ggc tcc ttc ttc ctctac agc aag ctc acc gtg gac aag agc agg 1410 Asp Gly Ser Phe Phe Leu TyrSer Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 420 tgg cag cag ggg aacgtc ttc tca tgc tcc gtg atg cat gag gct ctg 1458 Trp Gln Gln Gly Asn ValPhe Ser Cys Ser Val Met His Glu Ala Leu 425 430 435 cac aac cac tac acgcag aag agc ctc tcc ctg tct ccg ggt aaa 1503 His Asn His Tyr Thr Gln LysSer Leu Ser Leu Ser Pro Gly Lys 440 445 450 tgagtgccac ggccggcaagcccccgctcc ccaggctctc ggggtcgcgt gaggatgctt 1563 ggcacgtacc ccgtgtacatacttcccagg cacccagcat ggaaataaag cacccagcgc 1623 tgccctgggc ccctgcnaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1683 aaaaaaaaaa aatctctgcggccgc 1708 <210> SEQ ID NO 32 <211> LENGTH: 470 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 32 Met Glu Leu Gly Leu Ser TrpVal Phe Leu Val Ala Ile Leu Glu Gly 1 5 10 15 Val Gln Cys Glu Val GlnLeu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Asp Met HisTrp Val Arg Gln Ala Thr Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ala IleGly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly 65 70 75 80 Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser 85 90 95 Leu Tyr Leu Gln MetAsn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Val ArgAsp Asn Arg Lys Val Thr His Glu His Tyr Tyr Tyr 115 120 125 Tyr Gly MetAsp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ala SerThr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr GlnThr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys ValAsp Lys 225 230 235 240 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro ProCys Pro Ala Pro 245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe ProPro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu ValThr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val GlnPhe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Phe Asn 305 310 315 320 Ser Thr Phe Arg Val ValSer Val Leu Thr Val Val His Gln Asp Trp 325 330 335 Leu Asn Gly Lys GluTyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350 Ala Pro Ile GluLys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410415 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> SEQ ID NO 33 <211>LENGTH: 948 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: 5′UTR <222> LOCATION: (1)...(27) <221> NAME/KEY: CDS<222> LOCATION: (28)...(735) <221> NAME/KEY: 3′UTR <222> LOCATION:(739)...(948) <221> NAME/KEY: sig_peptide <222> LOCATION: (28)...(87)<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(948) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 33 gaattcgcag tgccatgacaccacgcc atg gaa acc cca gcg cag ctt ctc ttc 54 Met Glu Thr Pro Ala GlnLeu Leu Phe -20 -15 ctc ctg cta ctc tgg ctc cca gat acc acc gga gaa attgtg ttg acg 102 Leu Leu Leu Leu Trp Leu Pro Asp Thr Thr Gly Glu Ile ValLeu Thr -10 -5 1 5 cag tct cca ggc acc ctg tct ttg tct cca ggg gaa agagcc acc ctc 150 Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg AlaThr Leu 10 15 20 tcc tgc agg gcc agt cag aat att aga agc agc tac tta gcctgg tac 198 Ser Cys Arg Ala Ser Gln Asn Ile Arg Ser Ser Tyr Leu Ala TrpTyr 25 30 35 cag cag aaa cct ggc cag gct ccc ggg ctc ctc atc tat ggt gcatcc 246 Gln Gln Lys Pro Gly Gln Ala Pro Gly Leu Leu Ile Tyr Gly Ala Ser40 45 50 agc agg gcc act ggc atc cca gac agg ttc agt ggc agt ggg tct ggg294 Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 5560 65 aca gac ttc act ctc acc atc agc aga ctg gag cct gaa gat ttt gca342 Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala 7075 80 85 gtg tat tac tgt cag cag ttt ggt agc tca cct atg tgc agt ttt ggc390 Val Tyr Tyr Cys Gln Gln Phe Gly Ser Ser Pro Met Cys Ser Phe Gly 9095 100 cag ggg acc aag ctg gag atc aaa cga act gtg gct gca cca tct gtc438 Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val 105110 115 ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct486 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 120125 130 gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag534 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 135140 145 tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc582 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 150155 160 165 aca gag cag gac agc aag gac agc acc tac agc ctc agc agc accctg 630 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu170 175 180 acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc tgcgaa 678 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu185 190 195 gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc aacagg 726 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg200 205 210 gga gag tgt tagagggaga antgccccca cctgctcctc agttccagcc 775Gly Glu Cys 215 tgaccccctc ccatcctttg gcctctgacc ctttttccac aggggacctacccctattgc 835 ggtcctccag ctcatctttc acctcacccc cctcctcctc cttggctttaattatgctaa 895 tgttggagga gaatgaataa ataaagtgaa tctttgcacc tgtgaaaaaaaaa 948 <210> SEQ ID NO 34 <211> LENGTH: 236 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 34 Met Glu Thr Pro Ala Gln LeuLeu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu IleVal Leu Thr Gln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu ArgAla Thr Leu Ser Cys Arg Ala Ser Gln Asn 35 40 45 Ile Arg Ser Ser Tyr LeuAla Trp Tyr Gln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Gly Leu Leu Ile TyrGly Ala Ser Ser Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Arg Leu Glu ProGlu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe 100 105 110 Gly Ser Ser ProMet Cys Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg ThrVal Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu GlnLeu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly LeuSer 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230235 <210> SEQ ID NO 35 <211> LENGTH: 1673 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: 5′UTR <222>LOCATION: (1)...(95) <221> NAME/KEY: CDS <222> LOCATION: (96)...(1505)<221> NAME/KEY: 3′UTR <222> LOCATION: (1509)...(1673) <221> NAME/KEY:sig_peptide <222> LOCATION: (96)...(152) <400> SEQUENCE: 35 gaattcgcagtgccatgaca ccacggtgga gccccagcct tgggattccc aagtgtttgt 60 attcagtgatcaggactgaa cacacaggac tcacc atg gag ttg ggg ctg agc 113 Met Glu Leu GlyLeu Ser -15 tgg gtt ttc ctt gtt gct ata tta gaa ggt gtc cag tgt gag gtgcag 161 Trp Val Phe Leu Val Ala Ile Leu Glu Gly Val Gln Cys Glu Val Gln-10 -5 1 ctg gtg gag tct ggg gga ggc ttg gta cag cct ggg ggg tcc ctg aga209 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 5 1015 ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tac gac atg cac 257Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Asp Met His 20 25 3035 tgg gtc cgc caa gct aca gga aaa ggt ctg gag tgg gtc tca gct att 305Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val Ser Ala Ile 40 45 50ggt act gct ggt gac aca tac tat cca ggc tcc gtg aag ggc cga ttc 353 GlyThr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys Gly Arg Phe 55 60 65 accatc tcc aga gaa aat gcc aag aac tcc ttg tat ctt caa atg aac 401 Thr IleSer Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn 70 75 80 agc ctgaga gcc ggg gac acg gct gtg tat tac tgt gta aga gat aag 449 Ser Leu ArgAla Gly Asp Thr Ala Val Tyr Tyr Cys Val Arg Asp Lys 85 90 95 agg acg gtgacc cac gag cac tac tac tac tac ggt atg gac gtc tgg 497 Arg Thr Val ThrHis Glu His Tyr Tyr Tyr Tyr Gly Met Asp Val Trp 100 105 110 115 ggc caaggg acc acg gtc acc gtc tcc tca gcc tcc acc aag ggc cca 545 Gly Gln GlyThr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 120 125 130 tcg gtcttc ccc ctg gcg ccc tgc tcc agg agc acc tcc gag agc aca 593 Ser Val PhePro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 135 140 145 gcg gccctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acc 641 Ala Ala LeuGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 150 155 160 gtg tcgtgg aac tca ggc gct ctg acc agc ggc gtg cac acc ttc cca 689 Val Ser TrpAsn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 gct gtccta cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc 737 Ala Val LeuGln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 195 gtgccc tcc agc aac ttc ggc acc cag acc tac acc tgc aac gta gat 785 Val ProSer Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp 200 205 210 cacaag ccc agc aac acc aag gtg gac aag aca gtt gag cgc aaa tgt 833 His LysPro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys 215 220 225 tgtgtc gag tgc cca ccg tgc cca gca cca cct gtg gca gga ccg tca 881 Cys ValGlu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser 230 235 240 gtcttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg 929 Val PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 acccct gag gtc acg tgc gtg gtg gtg gac gtg agc cac gaa gac ccc 977 Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 275gag gtc cag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc 1025 GluVal Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 280 285 290aag aca aag cca cgg gag gag cag ttc aac agc acg ttc cgt gtg gtc 1073 LysThr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val 295 300 305agc gtc ctc acc gtt gtg cac cag gac tgg ctg aac ggc aag gag tac 1121 SerVal Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 310 315 320aag tgc aag gtc tcc aac aaa ggc ctc cca gcc ccc atc gag aaa acc 1169 LysCys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335atc tcc aaa acc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg 1217 IleSer Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350355 ccc cca tcc cgg gag gag atg acc aag aac cag gtc agc ctg acc tgc 1265Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 360 365370 ctg gtc aaa ggc ttc tac ccc agc gac atc gcc gtg gag tgg gag agc 1313Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 375 380385 aat ggg cag ccg gag aac aac tac aag acc aca cct ccc atg ctg gac 1361Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp 390 395400 tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc 1409Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410415 agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct 1457Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425430 435 ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa1505 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 440445 450 tgagtgccac ggccggcaag cccccgctcc ccaggctctc ggggtcgcgtgaggatgctt 1565 ggcacgtacc ccgtgtacat acttcccagg cacccagcat ggaaataaagcacccagcgc 1625 tgccctgggc ccctgcgaaa aaaaaaaaaa aaaaatctct gcggccgc1673 <210> SEQ ID NO 36 <211> LENGTH: 470 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 36 Met Glu Leu Gly Leu Ser TrpVal Phe Leu Val Ala Ile Leu Glu Gly 1 5 10 15 Val Gln Cys Glu Val GlnLeu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Asp Met HisTrp Val Arg Gln Ala Thr Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ala IleGly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly 65 70 75 80 Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser 85 90 95 Leu Tyr Leu Gln MetAsn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Val ArgAsp Lys Arg Thr Val Thr His Glu His Tyr Tyr Tyr 115 120 125 Tyr Gly MetAsp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140 Ala SerThr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 145 150 155 160Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr GlnThr 210 215 220 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys ValAsp Lys 225 230 235 240 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro ProCys Pro Ala Pro 245 250 255 Pro Val Ala Gly Pro Ser Val Phe Leu Phe ProPro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu ValThr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val GlnPhe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys ThrLys Pro Arg Glu Glu Gln Phe Asn 305 310 315 320 Ser Thr Phe Arg Val ValSer Val Leu Thr Val Val His Gln Asp Trp 325 330 335 Leu Asn Gly Lys GluTyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350 Ala Pro Ile GluLys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln ValTyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380 Gln ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410415 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 <210> SEQ ID NO 37 <211>LENGTH: 970 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: 5′UTR <222> LOCATION: (1)...(32) <221> NAME/KEY: CDS<222> LOCATION: (33)...(740) <221> NAME/KEY: 3′UTR <222> LOCATION:(744)...(970) <221> NAME/KEY: sig_peptide <222> LOCATION: (33)...(92)<400> SEQUENCE: 37 gaattcgcag tgccatgaca ccacggggaa cc atg gaa acc ccagcg cag ctt 53 Met Glu Thr Pro Ala Gln Leu -20 -15 ctc ttc ctc ctg ctactc tgg ctc cca gat acc acc gga gaa att gtg 101 Leu Phe Leu Leu Leu LeuTrp Leu Pro Asp Thr Thr Gly Glu Ile Val -10 -5 1 ttg acg cag tct cca ggcacc ctg tct ttg tct cca ggg gaa aga gcc 149 Leu Thr Gln Ser Pro Gly ThrLeu Ser Leu Ser Pro Gly Glu Arg Ala 5 10 15 acc ctc tcc tgc agg gcc agtcag agt att agc agc agc tcc tta gcc 197 Thr Leu Ser Cys Arg Ala Ser GlnSer Ile Ser Ser Ser Ser Leu Ala 20 25 30 35 tgg tac cag cag aaa cct ggccag gct ccc ggg ctc ctc atc ttt ggt 245 Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Gly Leu Leu Ile Phe Gly 40 45 50 gca tcc agc agg gcc act ggc atccca gac agg ttc agt ggc agt ggg 293 Ala Ser Ser Arg Ala Thr Gly Ile ProAsp Arg Phe Ser Gly Ser Gly 55 60 65 tct ggg aca gac ttc act ctc acc atcagc aga ctg gag cct gaa gat 341 Ser Gly Thr Asp Phe Thr Leu Thr Ile SerArg Leu Glu Pro Glu Asp 70 75 80 ttt gca gtg tat tac tgt cag cag ttt ggtagc tca cct atg tgc agt 389 Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly SerSer Pro Met Cys Ser 85 90 95 ttt ggc cag ggg acc aag ctg gag atc aaa cgaact gtg gct gca cca 437 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg ThrVal Ala Ala Pro 100 105 110 115 tct gtc ttc atc ttc ccg cca tct gat gagcag ttg aaa tct gga act 485 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu GlnLeu Lys Ser Gly Thr 120 125 130 gcc tct gtt gtg tgc ctg ctg aat aac ttctat ccc aga gag gcc aaa 533 Ala Ser Val Val Cys Leu Leu Asn Asn Phe TyrPro Arg Glu Ala Lys 135 140 145 gta cag tgg aag gtg gat aac gcc ctc caatcg ggt aac tcc cag gag 581 Val Gln Trp Lys Val Asp Asn Ala Leu Gln SerGly Asn Ser Gln Glu 150 155 160 agt gtc aca gag cag gac agc aag gac agcacc tac agc ctc agc agc 629 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser ThrTyr Ser Leu Ser Ser 165 170 175 acc ctg acg ctg agc aaa gca gac tac gagaaa cac aaa gtc tac gcc 677 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr Ala 180 185 190 195 tgc gaa gtc acc cat cag ggc ctg agctcg ccc gtc aca aag agc ttc 725 Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser Phe 200 205 210 aac agg gga gag tgt tagagggagaagtgccccca cctgctcctc agttccagcc 780 Asn Arg Gly Glu Cys 215 tgaccccctcccatcctttg gcctctgacc ctttttccac aggggaccta cccctattgc 840 ggtcctccagctcatctttc acctcacccc cctcctcctc cttggcttta attatgctaa 900 tgttggaggagaatgaataa ataaagtgaa tctttgcaaa aaaaaaaaaa aaaaaaatct 960 ctgcggccgc970 <210> SEQ ID NO 38 <211> LENGTH: 236 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <400> SEQUENCE: 38 Met Glu Thr Pro Ala Gln Leu Leu Phe LeuLeu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu ThrGln Ser Pro Gly Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr LeuSer Cys Arg Ala Ser Gln Ser 35 40 45 Ile Ser Ser Ser Ser Leu Ala Trp TyrGln Gln Lys Pro Gly Gln Ala 50 55 60 Pro Gly Leu Leu Ile Phe Gly Ala SerSer Arg Ala Thr Gly Ile Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Arg Leu Glu Pro Glu Asp PheAla Val Tyr Tyr Cys Gln Gln Phe 100 105 110 Gly Ser Ser Pro Met Cys SerPhe Gly Gln Gly Thr Lys Leu Glu Ile 115 120 125 Lys Arg Thr Val Ala AlaPro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys SerGly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr ProArg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln SerGly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 SerThr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210>SEQ ID NO 39 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Artificiallysynthesized primer sequence <221> NAME/KEY: primer_bind <222> LOCATION:(1)...(35) <400> SEQUENCE: 39 gaggtctccg ccctcgagat gcggctgggc agtcc 35<210> SEQ ID NO 40 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Artificiallysynthesized primer sequence <221> NAME/KEY: primer_bind <222> LOCATION:(1)...(33) <400> SEQUENCE: 40 cacaggacag ccaggggatc ccacgtggcc gcg 33<210> SEQ ID NO 41 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 41 Met Tyr Pro Pro Pro Tyr 1 5 <210> SEQ ID NO42 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 42 Tyr Met Asn Met 1 <210> SEQ ID NO 43 <211> LENGTH: 4 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 43 Tyr Val LysMet 1

What is claimed is:
 1. A human antibody which binds to AILIM.
 2. Thehuman antibody of claim 1, wherein said AILIM is derived from human. 3.A human monoclonal antibody which binds to AILIM or a portion thereof.4. The human monoclonal antibody or a portion thereof of claim 3,wherein said AILIM is derived from human.
 5. The human monoclonalantibody or a portion thereof of claim 3, wherein said human monoclonalantibody has an activity to inhibit a signal transduction into a cellmediated by AILIM.
 6. The human monoclonal antibody or a portion thereofof claim 5, wherein said activity to inhibit a signal transduction is(a) or (b) of the followings: (a) activity to inhibit proliferation ofAILIM-expressing cells, or (b) activity to inhibit cytokine productionfrom AILIM-expressing cells.
 7. The human monoclonal antibody or aportion thereof of claim 6, wherein said cytokine is one of thecytokines produced by Th1-type or Th2-type T cell.
 8. The humanmonoclonal antibody or a portion thereof of claim 7, wherein saidcytokine is interferon γ or interleukin
 4. 9. The human monoclonalantibody or a portion thereof of claim 5, wherein said human monoclonalantibody has an activity to prevent mixed lymphocyte reaction.
 10. Thehuman monoclonal antibody or a portion thereof of claim 3, wherein saidhuman monoclonal antibody has an activity to induce signal transductioninto a cell mediated by AILIM.
 11. The human monoclonal antibody or aportion thereof of claim 10, wherein said activity to induce signaltransduction is (a) or (b) of the followings: (a) activity to induceproliferation of AILIM-expressing cells, or (b) activity to inducecytokine production from AILIM-expressing cells.
 12. The humanmonoclonal antibody or a portion thereof of claim 11, wherein saidcytokine is one of the cytokines produced by Th1-type or Th2-type Tcell.
 13. The human monoclonal antibody or a portion thereof of claim12, wherein said cytokine is interferon γ or interleukin
 4. 14. Thehuman monoclonal antibody or a portion thereof of claim 3, wherein saidhuman monoclonal antibody has an activity to induce antibody-dependentcytotoxicity to AILIM-expressing cells, and/or immune cytolysis orapoptosis of AILIM-expressing cells.
 15. The human monoclonal antibodyor a portion thereof of claim 3, wherein the binding rate constant (ka)between said monoclonal antibody and AILIM is 1.0×10³ (1/M. Sec) ormore.
 16. The human monoclonal antibody or a portion thereof of claim15, wherein said binding rate constant (ka) is 1.0×10⁴ (1/M.Sec) ormore.
 17. The human monoclonal antibody or a portion thereof of claim16, wherein said binding rate constant (ka) is 1.0×10⁵ (1/M.Sec) ormore.
 18. The human monoclonal antibody or a portion thereof of claim 3,wherein the dissociation rate constant (kd) between said monoclonalantibody and AILIM is 1.0×10⁻³ (1/Sec) or less.
 19. The human monoclonalantibody or a portion thereof of claim 18, wherein said dissociationrate constant (kd) is 1.0×10⁻⁴ (1/Sec) or less.
 20. The human monoclonalantibody or a portion thereof of claim 19, wherein said dissociationrate constant (kd) is 1.0×10⁻⁵ (1/Sec) or less.
 21. The human monoclonalantibody or a portion thereof of claim 3, wherein the dissociationconstant (Kd) between said monoclonal antibody and AILIM is 1.0×10⁻⁶ (M)or less.
 22. The human monoclonal antibody or a portion thereof of claim21, wherein said dissociation constant (Kd) is 1.0×10⁻⁷ (M) or less. 23.The human monoclonal antibody or a portion thereof of claim 22, whereinsaid dissociation constant (Kd) is 1.0×10⁻⁸ (M) or less.
 24. The humanmonoclonal antibody or a portion thereof of claim 23, wherein saiddissociation constant (Kd) is 1.0×10⁻⁹ (M) or less.
 25. The humanmonoclonal antibody or a portion thereof of claim 4, wherein a V regionDNA encoding a heavy chain variable region of said human monoclonalantibody is derived from either the human immunoglobulin heavy chain Vgene segment 1-02 or 3-13.
 26. The human monoclonal antibody or aportion thereof of claim 4, wherein a V region DNA encoding a lightchain variable region of said human monoclonal antibody is derived fromeither the human immunoglobulin light chain V gene segment L5 or A27.27. The human monoclonal antibody or a portion thereof of claim 25,wherein a V region DNA encoding a heavy chain variable region of saidhuman monoclonal antibody is derived from either the humanimmunoglobulin heavy chain V gene segment 1-02 or 3-13, and wherein a Vregion DNA encoding a light chain variable region of said humanmonoclonal antibody is derived from either the human immunoglobulinlight chain V gene segment L5 or A27.
 28. The human monoclonal antibodyor a portion thereof of claim 27, wherein the V region DNA encoding aheavy chain variable region of said human monoclonal antibody is derivedfrom the human immunoglobulin heavy chain V gene segment 1-02, and the Vregion DNA encoding a light chain variable region of said humanmonoclonal antibody is derived from the human immunoglobulin light chainV gene segment L5.
 29. The human monoclonal antibody or a portionthereof of claim 27, wherein the V region DNA encoding a heavy chainvariable region of said human monoclonal antibody is derived from thehuman immunoglobulin heavy chain V gene segment 3-13, and the V regionDNA encoding a light chain variable region of said human monoclonalantibody is derived from the human immunoglobulin light chain V genesegment A27.
 30. The human monoclonal antibody or a portion thereof ofclaim 4, wherein a heavy chain variable region of said human monoclonalantibody has an amino acid sequence defined in any of the following (a)through (f): (a) amino acid sequence comprising amino acids fromposition 20 through 117 of SEQ ID NO:28, (b) amino acid sequencecomprising amino acids from position 20 through 117 of SEQ ID NO:28 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (c) aminoacid sequence comprising amino acids from position 20 through 116 of SEQID NO:32, (d) amino acid sequence comprising amino acids from position20 through 116 of SEQ ID NO:32 in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added. (e) amino acid sequence comprising amino acidsfrom position 20 through 116 of SEQ ID NO:36, or (f) amino acid sequencecomprising amino acids from position 20 through 116 of SEQ ID NO:36, inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added.
 31. Thehuman monoclonal antibody or a portion thereof of claim 4, wherein aheavy chain polypeptide of said human monoclonal antibody has an aminoacid sequence defined in any of the following (a) through (f): (a) aminoacid sequence comprising amino acids from position 20 through 470 of SEQID NO:28, (b) amino acid sequence comprising amino acids from position20 through 470 of SEQ ID NO:28 in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added. (c) amino acid sequence comprising amino acidsfrom position 20 through 470 of SEQ ID NO:32, (d) amino acid sequencecomprising amino acids from position 20 through 470 of SEQ ID NO:32 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (e) aminoacid sequence comprising amino acids from position 20 through 470 of SEQID NO:36, or (f) amino acid sequence comprising amino acids fromposition 20 through 470 of SEQ ID NO:36 in which one or more amino acidresidues are deleted or substituted, or to which one or more amino acidresidues are inserted or added.
 32. The human monoclonal antibody or aportion thereof of claim 4, wherein a light chain variable region ofsaid human monoclonal antibody has an amino acid sequence defined in anyof the following (a) through (f): (a) amino acid sequence comprisingamino acids from position 23 through 116 of SEQ ID NO:30, (b) amino acidsequence comprising amino acids from position 23 through 116 of SEQ IDNO:30 in which one or more amino acid residues are deleted orsubstituted, or to which one or more amino acid residues are inserted oradded. (c) amino acid sequence comprising amino acids from position 21through 116 of SEQ ID NO:34, (d) amino acid sequence comprising aminoacids from position 21 through 116 of SEQ ID NO:34 in which one or moreamino acid residues are deleted or substituted, or to which one or moreamino acid residues are inserted or added. (e) amino acid sequencecomprising amino acids from position 21 through 116 of SEQ ID NO:38, or(f) amino acid sequence comprising amino acids from position 21 through116 of SEQ ID NO:38 in which one or more amino acid residues are deletedor substituted, or to which one or more amino acid residues are insertedor added.
 33. The human monoclonal antibody or a portion thereof ofclaim 4, wherein a light chain polypeptide of said human monoclonalantibody has an amino acid sequence defined in any of the following (a)through (f): (a) amino acid sequence comprising amino acids fromposition 23 through 236 of SEQ ID NO:30, (b) amino acid sequencecomprising amino acids from position 23 through 236 of SEQ ID NO:30 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added. (c) aminoacid sequence comprising amino acids from position 21 through 236 of SEQID NO:34, (d) amino acid sequence comprising amino acids from position21 through 236 of SEQ ID NO:34 in which one or more amino acid residuesare deleted or substituted, or to which one or more amino acid residuesare inserted or added. (e) amino acid sequence comprising amino acidsfrom position 21 through 236 of SEQ ID NO:38, or (f) amino acid sequencecomprising amino acids from position 21 through 236 of SEQ ID NO:38 inwhich one or more amino acid residues are deleted or substituted, or towhich one or more amino acid residues are inserted or added.
 34. Thehuman monoclonal antibody or a portion thereof of claim 4, wherein saidhuman monoclonal antibody has the following characteristics (a) and (b):(a) a heavy chain variable region has an amino acid sequence comprisingthe amino acid sequence from amino acid 20 through 117 according to SEQID NO:28, and (b) a light chain variable region has an amino acidsequence comprising the amino acid sequence from amino acid 23 through116 according to SEQ ID NO:30.
 35. The human monoclonal antibody or aportion thereof of claim 4, wherein said human monoclonal antibody hasthe following characteristics (a) and (b): (a) a heavy chain polypeptidehas an amino acid sequence from amino acid 20 through 470 according toSEQ ID NO:28, and (b) a light chain polypeptide has an amino acidsequence from amino acid 23 through 236 according to SEQ ID NO:30. 36.The human monoclonal antibody or a portion thereof of claim 4, whereinsaid human monoclonal antibody has the following characteristics (a) and(b): (a) a heavy chain variable region has an amino acid sequencecomprising the amino acid sequence from amino acid 20 through 116according to SEQ ID NO:32, and (b) a light chain variable region has anamino acid sequence comprising the amino acid sequence from amino acid21 through 116 according to SEQ ID NO:34.
 37. The human monoclonalantibody or a portion thereof of claim 4, wherein said human monoclonalantibody has the following characteristics (a) and (b): (a) a heavychain polypeptide has an amino acid sequence comprising the amino acidsequence from amino acid 20 through 470 according to SEQ ID NO:32, and(b) a light chain polypeptide has an amino acid sequence comprising theamino acid sequence from amino acid 21 through 236 according to SEQ IDNO:34.
 38. The human monoclonal antibody or a portion thereof of claim4, wherein said human monoclonal antibody has the followingcharacteristics (a) and (b): (a) a heavy chain variable region has anamino acid sequence comprising the amino acid sequence from amino acid20 through 116 according to SEQ ID NO:36, and (b) a light chain variableregion has an amino acid sequence comprising the amino acid sequencefrom amino acid 21 through 116 according to SEQ ID NO:38.
 39. The humanmonoclonal antibody or a portion thereof of claim 4, wherein said humanmonoclonal antibody has the following characteristics (a) and (b): (a) aheavy chain polypeptide has an amino acid sequence comprising the aminoacid sequence from amino acid 20 through 470 according to SEQ ID NO:36,and (b) a light chain polypeptide has an amino acid sequence comprisingthe amino acid sequence from amino acid 21 through 236 according to SEQID NO:38.
 40. The human monoclonal antibody or a portion thereof ofclaim 3, wherein said human monoclonal antibody is a monoclonal antibodyderived from a transgenic non-human mammal capable of producing humanantibodies.
 41. The human monoclonal antibody or a portion thereof ofclaim 40, wherein said human monoclonal antibody is obtained byimmunizing transgenic non-human mammal capable of producing humanantibody with AILIM-expressing cells, membrane fractions derived fromsaid cells, whole molecules constituting AILIM or a portion thereof, orgenes encoding AILIM or a portion thereof.
 42. The human monoclonalantibody or a portion thereof of claim 40, wherein said transgenicnon-human mammal is a transgenic mouse.
 43. A DNA or a portion thereofencoding a polypeptide selected from the group consisting of (a) through(f) below: (a) a polypeptide comprising the amino acid sequence fromamino acid 20 through 117 according to SEQ ID NO:28, (b) a polypeptidecomprising the amino acid sequence from amino acid 23 through 116according to SEQ ID NO:30, (c) a polypeptide comprising the amino acidsequence from amino acid 20 through 116 according to SEQ ID NO:32, (d) apolypeptide comprising the amino acid sequence from amino acid 21through 116 according to SEQ ID NO:34, (e) a polypeptide comprising theamino acid sequence from amino acid 20 through 116 according to SEQ IDNO:36, and (f) a polypeptide comprising the amino acid sequence fromamino acid 21 through 116 according to SEQ ID NO:38.
 44. A DNA or aportion thereof encoding a polypeptide selected from the groupconsisting of (a) through (f) below: (a) a polypeptide comprising theamino acid sequence from amino acids 20 through 470 according to SEQ IDNO:28, (b) a polypeptide comprising the amino acid sequence from aminoacids 23 through 236 according to SEQ ID NO:30, (c) a polypeptidecomprising the amino acid sequence from amino acids 20 through 470according to SEQ ID NO:32, (d) a polypeptide comprising the amino acidsequence from amino acids 21 through 236 according to SEQ ID NO:34, (e)a polypeptide comprising the amino acid sequence from amino acids 20through 470 according to SEQ ID NO:36, and (f) a polypeptide comprisingthe amino acid sequence from amino acids 21 through 236 according to SEQID NO:38.
 45. A DNA or a portion thereof selected from the groupconsisting of (a) through (f) below: (a) a DNA comprising the nucleotidesequence from nucleotides 126 through 419 according to SEQ ID NO:27, (b)a DNA comprising the nucleotide sequence from nucleotides 105 through386 according to SEQ ID NO:29, (c) a DNA comprising the nucleotidesequence from nucleotides 151 through 441 according to SEQ ID NO:31, (d)a DNA comprising the nucleotide sequence from nucleotides 88 through 375according to SEQ ID NO:33, (e) a DNA comprising the nucleotide sequencefrom nucleotides 153 through 443 according to SEQ ID NO:35, and (f) aDNA comprising the nucleotide sequence from nucleotides 93 through 380according to SEQ ID NO:37.
 46. A DNA or a portion thereof selected froma group consisting of (a) through (f) below: (a) a DNA comprising thenucleotide sequence from nucleotides 69 through 1481 according to SEQ IDNO:27, (b) a DNA comprising the nucleotide sequence from nucleotides 39through 749 according to SEQ ID NO:29, (c) a DNA comprising thenucleotide sequence from nucleotides 94 through 1506 defined in SEQ IDNO:31, (d) a DNA comprising the nucleotide sequence from nucleotides 28through 738 according to SEQ ID NO:33, (e) a DNA comprising thenucleotide sequence from nucleotides 96 through 1508 according to SEQ IDNO:35, and (f) a DNA comprising the nucleotide sequence from nucleotides33 through 743 according to SEQ ID NO:37.
 47. A vector comprising theDNA of claim
 43. 48. The vector of claim 47 comprising a DNA accordingto any of the following (a) through (c): (a) a DNA comprising thenucleotide sequence from nucleotides 126 through 419 according to SEQ IDNO:27, (b) a DNA comprising the nucleotide sequence from nucleotides 151through 441 according to SEQ ID NO:31, or (c) a DNA comprising thenucleotide sequence from nucleotides 153 through 443 according to SEQ IDNO:35.
 49. The vector of claim 47 comprising a DNA according to any ofthe following (a) through (c): (a) a DNA comprising the nucleotidesequence from nucleotides 69 through 1481 according to SEQ ID NO:27, (b)a DNA comprising the nucleotide sequence from nucleotides 94 through1506 according to SEQ ID NO:31, or (c) a DNA comprising the nucleotidesequence from nucleotides 96 through 1508 according to SEQ ID NO:35. 50.The vector of claim 47 comprising a DNA according to any of thefollowing (a) through (c): (a) a DNA comprising the nucleotide sequencefrom nucleotides 105 through 386 according to SEQ ID NO:29, (b) a DNAcomprising the nucleotide sequence from nucleotides 88 through 375according to SEQ ID NO:33, or (c) a DNA comprising the nucleotidesequence from nucleotides 93 through 380 according to SEQ ID NO:37. 51.The vector of claim 47 comprising a DNA according to any of thefollowing (a) through (c): (a) a DNA comprising the nucleotide sequencefrom nucleotides 39 through 749 according to SEQ ID NO:29, (b) a DNAcomprising the nucleotide sequence from nucleotides 28 through 738according to SEQ ID NO:33, or (c) a DNA comprising the nucleotidesequence from nucleotides 33 through 743 according to SEQ ID NO:37. 52.The vector of claim 47 comprising a DNA according to the following (a)and(b): (a) a DNA comprising the nucleotide sequence from nucleotides126 through 419 according to SEQ ID NO:27, and (b) a DNA comprising thenucleotide sequence from nucleotides 105 through 386 according to SEQ IDNO:29.
 53. The vector of claim 47 comprising a DNA according to thefollowing (a) and (b): (a) a DNA comprising the nucleotide sequence fromnucleotides 69 through 1481 according to SEQ ID NO:27, and (b) a DNAcomprising the nucleotide sequence from nucleotides 39 through 749according to SEQ ID NO:29.
 54. The vector of claim 47 comprising a DNAaccording to the following (a) and (b): (a) a DNA comprising thenucleotide sequence from nucleotides 151 through 441 according to SEQ IDNO:31, and (b) a DNA comprising the nucleotide sequence from nucleotides88 through 375 according to SEQ ID NO:33.
 55. The vector of claim 47comprising a DNA according to the following (a) and (b): (a) a DNAcomprising the nucleotide sequence from nucleotides 94 through 1506according to SEQ ID NO:31, and (b) a DNA comprising the nucleotidesequence from nucleotides 28 through 738 according to SEQ ID NO:33. 56.The vector of claim 47 comprising a DNA according to the following (a)and (b): (a) a DNA comprising the nucleotide sequence from nucleotides153 through 443 according to SEQ ID NO:35, and (b) a DNA comprising thenucleotide sequence from nucleotides 93 through 380 according to SEQ IDNO:37.
 57. The vector of claim 47 comprising a DNA according to thefollowing (a) and (b): (a) a DNA comprising the nucleotide sequence fromnucleotides 96 through 1508 according to SEQ ID NO:35, and (b) a DNAcomprising the nucleotide sequence from nucleotides 33 through 743according to SEQ ID NO:37.
 58. A cell producing a human monoclonalantibody of claim
 3. 59. The cell of claim 58, wherein said cell is afused cell obtained by fusing B cell, derived from a mammal capable ofproducing said human monoclonal antibody, and myeloma cell derived froma mammal.
 60. A genetic recombinant host transformed by transferring aDNA described below in (a) or a vector comprising said DNA, a DNAdescribed below in (b) or a vector comprising said DNA, or both DNAsdescribed below in (a) and (b) or a vector comprising both of said DNAs:(a) a DNA encoding a heavy chain polypeptide or a portion thereof of amonoclonal antibody which binds to human AILIM; or (b) a DNA encoding alight chain polypeptide or a portion thereof of a monoclonal antibodywhich binds to human AILIM.
 61. The genetic recombinant host of claim60, wherein said monoclonal antibody is a human monoclonal antibody. 62.The genetic recombinant host of claim 60, wherein said host is amammalian cell.
 63. The genetic recombinant host of claim 60, whereinsaid host is a mammalian fertilized egg.
 64. The genetic recombinanthost of claim 60, wherein said heavy chain polypeptide is one of theheavy chain polypeptides selected from the group consisting of thefollowing (a) through (c): (a) a heavy chain polypeptide comprising theamino acid sequence from amino acids 20 through 117 according to SEQ IDNO:28, (b) a heavy chain polypeptide comprising the amino acid sequencefrom amino acids 20 through 116 according to SEQ ID NO:32, and (c) aheavy chain polypeptide comprising the amino acid sequence from aminoacids 20 through 116 according to SEQ ID NO:36.
 65. The geneticrecombinant host of claim 60, wherein said heavy chain polypeptide isone of the heavy chain polypeptide selected from the group consisting ofthe following (a) through (c): (a) a heavy chain polypeptide comprisingthe amino acid sequence from amino acids 20 through 470 according to SEQID NO:28, (b) a heavy chain polypeptide comprising the amino acidsequence from amino acids 20 through 470 according to SEQ ID NO:32, and(c) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 20 through 470 according to SEQ ID NO:36.
 66. The geneticrecombinant host of claim 60, wherein said light chain polypeptide isone of the light chain polypeptide selected from the group consisting ofthe following (a) through (c): (a) a heavy chain polypeptide comprisingthe amino acid sequence from amino acids 23 through 116 according to SEQID NO:30, (b) a heavy chain polypeptide comprising the amino acidsequence from amino acids 21 through 116 according to SEQ ID NO:34, and(c) a heavy chain polypeptide comprising the amino acid sequence fromamino acids 21 through 116 according to SEQ ID NO:38.
 67. The geneticrecombinant host of claim 60, wherein said light chain polypeptide isone of the light chain polypeptide selected from the group consisting ofthe following (a) through (c): (a) a light chain polypeptide comprisingthe amino acid sequence from amino acids 23 through 236 according to SEQID NO:30, (b) a light chain polypeptide comprising the amino acidsequence from amino acids 21 through 236 according to SEQ ID NO:34, and(c) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 236 according to SEQ ID NO:38.
 68. The geneticrecombinant host of claim 60, wherein said heavy chain and light chainpolypeptides are those defined below in (a) and (b), respectively: (a) aheavy chain polypeptide comprising the amino acid sequence from aminoacids 20 through 117 according to SEQ ID NO:28, and (b) a light chainpolypeptide comprising the amino acid sequence from amino acids 23through 116 according to SEQ ID NO:30.
 69. The genetic recombinant hostof claim 60, wherein said heavy chain and light chain polypeptides arethose defined below in (a) and (b), respectively: (a) a heavy chainpolypeptide comprising the amino acid sequence from amino acids 20through 470 according to SEQ ID NO:28, and (b) a light chain polypeptidecomprising the amino acid sequence from amino acids 23 through 236according to SEQ ID NO:30.
 70. The genetic recombinant host of claim 60,wherein said heavy chain and light chain polypeptides are those definedbelow in (a) and (b), respectively: (a) a heavy chain polypeptidecomprising the amino acid sequence from amino acids 20 through 116according to SEQ ID NO:32, and (b) a light chain polypeptide comprisingthe amino acid sequence from amino acids 21 through 116 according to SEQID NO:34.
 71. The genetic recombinant host of claim 60, wherein saidheavy chain and light chain polypeptides are those defined below in (a)and (b), respectively: (a) a heavy chain polypeptide comprising theamino acid sequence from amino acids 20 through 470 according to SEQ IDNO:32, and (b) a light chain polypeptide comprising the amino acidsequence from amino acids 21 through 236 according to SEQ ID NO:34. 72.The genetic recombinant host of claim 60, wherein said heavy chain andlight chain polypeptides are those defined below in (a) and (b),respectively: (a) a heavy chain polypeptide comprising the amino acidsequence from amino acids 20 through 116 according to SEQ ID NO:36, and(b) a light chain polypeptide comprising the amino acid sequence fromamino acids 21 through 116 according to SEQ ID NO:38.
 73. The geneticrecombinant host of claim 60, wherein said heavy chain and light chainpolypeptides are those defined below in (a) and (b), respectively: (a) aheavy chain polypeptide comprising the amino acid sequence from aminoacids 20 through 470 according to SEQ ID NO:36, and (b) a light chainpolypeptide comprising the amino acid sequence from amino acids 21through 236 according to SEQ ID NO:38.
 74. The genetic recombinant hostof claim 60, wherein the DNA encoding said heavy chain polypeptide is aDNA defined in any of following (a) through (c): (a) a DNA comprisingthe nucleotide sequence from nucleotides 126 through 419 according toSEQ ID NO:27, (b) a DNA comprising the nucleotide sequence fromnucleotides 151 through 441 according to SEQ ID NO:31, and (c) a DNAcomprising the nucleotide sequence from nucleotides 153 through 443according to SEQ ID NO:35.
 75. The genetic recombinant host of claim 60,wherein the DNA encoding said heavy chain polypeptide is a DNA definedin any of following (a) through (c): (a) a DNA comprising the nucleotidesequence from nucleotides 69 through 1481 according to SEQ ID NO:27, (b)a DNA comprising the nucleotide sequence from nucleotides 94 through1506 according to SEQ ID NO:31, and (c) a DNA comprising the nucleotidesequence from nucleotides 96 through 1508 according to SEQ ID NO:35. 76.The genetic recombinant host of claim 60, wherein the DNA encoding saidlight chain polypeptide is a DNA defined in any of following (a) through(c): (a) a DNA comprising the nucleotide sequence from nucleotides 105through 386 according to SEQ ID NO:29, (b) a DNA comprising thenucleotide sequence from nucleotides 88 through 375 according to SEQ IDNO:33, and (c) a DNA comprising the nucleotide sequence from nucleotides93 through 380 according to SEQ ID NO:37.
 77. The genetic recombinanthost of claim 60, wherein the DNA encoding said light chain polypeptideis a DNA as defined in any of following (a) through (c): (a) a DNAcomprising the nucleotide sequence from nucleotides 39 through 749according to SEQ ID NO:29, (b) a DNA comprising the nucleotide sequencefrom nucleotides 28 through 738 according to SEQ ID NO:33, and (c) a DNAcomprising the nucleotide sequence from nucleotides 33 through 743according to SEQ ID NO:37.
 78. The genetic recombinant host of claim 60,wherein the DNA encoding said heavy chain polypeptide is a DNA describedbelow in (a), and the DNA encoding said light chain polypeptide is a DNAas described below in (b): (a) a DNA comprising the nucleotide sequencefrom nucleotides 126 through 419 according to SEQ ID NO:27, and (b) aDNA comprising the nucleotide sequence from nucleotides 105 through 386according to SEQ ID NO:29.
 79. The genetic recombinant host of claim 60,wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 69 through 1481 according to SEQ IDNO:27, and (b) a DNA comprising the nucleotide sequence from nucleotides39 through 749 according to SEQ ID NO:29.
 80. The genetic recombinanthost of claim 60, wherein the DNA encoding said heavy chain polypeptideis the DNA described below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 151 through 441 according to SEQ IDNO:31, and (b) a DNA comprising the nucleotide sequence from nucleotides88 through 375 V SEQ ID NO:33.
 81. The genetic recombinant host of claim60, wherein the DNA encoding said heavy chain polypeptide is the DNAdescribed below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 94 through 1506 according to SEQ IDNO:31, and (b) a DNA comprising the nucleotide sequence from nucleotides28 through 738 according to SEQ ID NO:33.
 82. The genetic recombinanthost of claim 60, wherein the DNA encoding said heavy chain polypeptideis the DNA described below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 153 through 443 according to SEQ IDNO:35, and (b) a DNA comprising the nucleotide sequence from nucleotides93 through 380 according to SEQ ID NO:37.
 83. The genetic recombinanthost of claim 60, wherein the DNA encoding said heavy chain polypeptideis the DNA described below in (a), and the DNA encoding said light chainpolypeptide is the DNA described below in (b): (a) a DNA comprising thenucleotide sequence from nucleotides 96 through 1508 according to SEQ IDNO:35, and (b) a DNA comprising the nucleotide sequence from nucleotides33 through 743 according to SEQ ID NO:37.
 84. A human monoclonalantibody or a portion thereof produced by a genetic recombinant host(provided excluding the case where said host is a fertilized egg) ofclaim
 60. 85. A pharmaceutical composition comprising the human antibodyof claim 1, and a pharmaceutically acceptable carrier.
 86. Apharmaceutical composition comprising the human monoclonal antibody or aportion thereof of claim 3, and a pharmaceutically acceptable carrier.87. A pharmaceutical composition comprising a human monoclonal antibodyor a portion thereof of claim 84, and a pharmaceutically acceptablecarrier.
 88. The pharmaceutical composition of claim 85, wherein saidpharmaceutical composition is used to inhibit signal transduction intothe cell mediated by AILIM.
 89. The pharmaceutical composition of claim85, wherein said pharmaceutical composition is used to preventproliferation of AILIM-expressing cells.
 90. The pharmaceuticalcomposition of claim 85, wherein said pharmaceutical composition is usedto prevent production of a cytokine from AILIM-expressing cells.
 91. Thepharmaceutical composition of claim 85, wherein said pharmaceuticalcomposition is used to induce signal transduction into a cell mediatedby AILIM.
 92. The pharmaceutical composition of claim 85, wherein saidpharmaceutical composition is used to induce proliferation ofAILIM-expressing cells.
 93. The pharmaceutical composition of claim 85,wherein said pharmaceutical composition is used to induce production ofa cytokine from AILIM-expressing cells.
 94. The pharmaceuticalcomposition of claim 85, wherein said pharmaceutical composition is usedto induce antibody-dependent cytotoxicity against AILIM-expressingcells, and/or immune cytolysis or apoptosis of AILIM-expressing cells.95. A pharmaceutical composition for preventing, treating, orprophylaxis of delayed type allergy, comprising a substance having anactivity in modulating signal transduction mediated by AILIM, and apharmaceutically acceptable carrier.
 96. The pharmaceutical compositionof claim 95, wherein the substance is a protein substance.
 97. Thepharmaceutical composition of claim 96, wherein the protein substance isselected from the group consisting of: a) an antibody which binds toAILIM or a portion thereof; b) a polypeptide comprising the whole or aportion of an extracellular region of AILIM; c) a fusion polypeptidecomprising the whole or a portion of an extracellular region of AILIM,and the whole or a portion of a constant region of immunoglobulin heavychain; and d) a polypeptide which binds to AILIM.
 98. The pharmaceuticalcomposition of claim 97, wherein said antibody that binds to AILIM is ahuman antibody.
 99. The pharmaceutical composition of claim 97, whereinsaid antibody that binds to AILIM is a human monoclonal antibody. 100.The pharmaceutical composition of claim 97, wherein said antibodyagainst AILIM is a human monoclonal antibody produced by a geneticrecombinant host transformed by transferring a DNA described below in(a) or a vector comprising said DNA, a DNA described below in (b) or avector comprising said DNA, or both DNAs described below in (a) and (b)or a vector comprising both of said DNAs: (a) a DNA encoding a heavychain polypeptide or a portion thereof of a monoclonal antibody whichbinds to human AILIM; or (b) a DNA encoding a light chain polypeptide ora portion thereof of a monoclonal antibody which binds to human AILIM.101. The pharmaceutical composition of claim 95, wherein the substanceis a non-protein substance.
 102. The pharmaceutical composition of claim101, wherein the non-protein substance is DNA, RNA, or a chemicallysynthesized compound.
 103. A method for identifying substances that bindto AILIM or AILIM ligand comprising the following processes: (a)preparing an insoluble carrier on which the entire extracellular regionof AILIM or a portion thereof is immobilized; (b) preparing apolypeptide comprising the whole extracellular region of AILIM ligand ora portion thereof labeled with a labeling material that emit adetectable signal; (c) reacting the insoluble carrier in process(a) withthe polypeptide in process (b); (d) reacting the insoluble carrier ofprocess (a), the polypeptide of process (b) and said substance to eachother in any arbitrary orders; (e) detecting the signal emitted fromsaid labeling material contained in the complex produced in process (c),and the signal emitted from said labeling material contained in thecomplex produced in process (d), respectively; and (f) comparing themagnitude of each of signals detected in process (e).
 104. A method foridentifying substances that bind to AILIM or AILIM ligand comprising thefollowing processes: (a) preparing an insoluble carrier on which theentire extracellular region of AILIM ligand or a portion thereof isimmobilized; (b) preparing a polypeptide comprising the wholeextracellular region of AILIM or a portion thereof labeled with alabeling material that emit a detectable signal; (c) reacting theinsoluble carrier in process (a) with the polypeptide in process (b);(d) reacting the insoluble carrier of process (a), the polypeptide ofprocess (b) and said substance to each other in any arbitrary orders;(e) detecting the signal emitted from said labeling material containedin the complex produced in process (c), and the signal emitted from saidlabeling material contained in the complex produced in process (d),respectively; and (f) comparing the magnitude of each of signalsdetected in process (e).
 105. The method of claim 103, wherein saidpolypeptide comprising the whole extracellular region of AILIM or aportion thereof is a fusion polypeptide comprising a polypeptide,comprising the whole extracellular region of AILIM or a portion thereof,and the whole constant region of immunoglobulin heavy chain or a portionthereof.
 106. The method of claim 103, wherein said polypeptidecomprising the whole extracellular region of AILIM ligand or a portionthereof is a fusion polypeptide comprising a polypeptide, comprising thewhole extracellular region of AILIM ligand or a portion thereof, and thewhole constant region of immunoglobulin heavy chain or a portionthereof.
 107. The method of claim 103, wherein said AILIM is a humanAILIM.
 108. The method of claim 103, wherein said AILIM ligand is ahuman AILIM ligand.